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The Binding of a Glycoprotein 120 V3 Loop Peptide to HIV-1 Neutralizing Antibodies

The structural and antigenic properties of a peptide (“CRK”) derived from the V3 loop of HIV-1 gp120 protein were studied using NMR and SPR techniques. The sequence of CRK corresponds to the central portion of the V3 loop containing the highly conserved “GPGR” residue sequence. Although the biological significance of this conserved sequence is unknown, the adoption of conserved secondary structure (type II β-turn) in this region has been proposed. The tendency of CRK (while free or conjugated to protein), to adopt such structure and the influence of such structure upon CRK antigenicity were investigated by NMR and SPR, respectively. Regardless of conjugation, CRK is conformationally averaged in solution but a weak tendency of the CRK “GPGR” residues to adopt a β-turn conformation was observed after conjugation. The influence of GPGR structure upon CRK antigenicity was investigated by measuring the affinities of two cognate antibodies: “5023A” and “5025A,” for CRK, protein-conjugated CRK and gp120 protein. Each antibody bound to all the antigens with nearly the same affinity. From these data, it appears that: (a) antibody binding most likely involves an induced fit of the peptide and (b) the gp120 V3 loop is probably conformationally heterogeneous. Since 5023A and 5025A are HIV-1 neutralizing antibodies, neutralization in these cases appears to be independent of adopted GPGR β-turn structure.

immunogens to elicit antibodies that possess HIV-1 virus neutralization capabilities (2,3). The binding of neutralizing antibodies blocks virus entry into the host cell but does not prevent binding of HIV-1 to its primary cell receptor protein, CD4 (4,5). Although the V3 loop represents an important target for the development of vaccines against AIDS, its high sequence variability also makes it a very problematic one (6). Nonetheless, the occurrence of the highly conserved residue sequence, "GPGR," at the tip of the V3 loop has raised the possibility that these residues make up a conserved secondary structural element in gp120. The function of such a structural element, if one exists, is presently unknown, however. The precise predicted secondary structure adopted by the GPGR residues is a type II ␤-turn (6,7).
In order to determine whether the conserved GPGR sequence confers certain secondary structural tendencies upon this region of the V3 loop, numerous NMR studies were conducted upon a variety of V3 loop-derived peptides (8 -21). These peptides were shown to have only low density populations of folded structure and to be conformationally averaged in solution. Despite the report of a "core" gp120 protein complex crystal structure, this complex was prepared using a form of gp120 which lacked most of the hypervariable loops including V3 (22). Information regarding the actual three-dimensional structure of the V3 loop in native gp120 is therefore still unavailable.
Due to the linear and flexible nature of V3 loop peptides in solution, a variety of methods have been employed to induce greater and presumably more "native" structure in previously studied V3 loop peptides. These methods included aminoisobutyric acid substitution (10), insertion into a viral coat protein (23), glycosylation (12)(13)(14), attachment to resin beads (24), cyclization of the peptide (15), and trifluoroethanol addition (14 -19).
One particular method used previously to induce structure in short, linear peptides involves covalent attachment (or "conjugation") of the peptide to BPTI protein. Using this method, a nine-residue peptide derived from hemagglutinin (25) was shown to significantly affect the solution structure of this peptide after its conjugation to BPTI (26). Due to the well characterized NMR properties of BPTI protein (27)(28)(29)(30)(31)(32) and the fact that no modification of the peptide or solvent conditions are required, we chose to employ this method in an attempt to induce greater CRK peptide structure for these solution NMR studies.
We began NMR and surface plasmon resonance (SPR) studies of a PND peptide in order to investigate the structural propensities of this peptide (while free and conjugated to BPTI), and their potential importance to the binding of this peptide by HIV-1 neutralizing antibodies. The PND peptide of interest, "RK," has the following sequence: Arg 1 -Ile 2 -Gln 3 -Arg 4 -Gly 5 -Pro 6 -Gly 7 -Arg 8 -Ala 9 -Phe 10 -Val 11 -Thr 12 -Ile 13 -Gly 14 -Lys 15 . This sequence, corresponding to residues 308 -322 of the gp120 envelope protein of HIV-1 (using the gp120 numbering scheme for strain IIIB, Ref. 33), comprises the tip of the gp120 V3 loop and represents the center of the HIV-1 PND. The two antibodies studied, "5023A" and "5025A" were both raised against RK and both exhibit HIV-1 virus neutralization as well as cell fusion inhibition capabilities in vitro (33). Since a cysteinylated peptide was needed for the protein conjugation study, the free and BPTI-conjugated form of an N-terminal cysteinylated form of RK ("CRK") were actually studied and compared by NMR techniques.
To determine the overall importance of folded CRK structure to its binding by its cognate antibodies, 5023A and 5025A, SPR techniques were used to measure the binding of these antibodies to various forms of the CRK antigen-free CRK peptide, BPTI-conjugated CRK peptide, and intact gp120. Based upon the NMR and SPR data presented in this study, the relationship between peptide antigenic structure versus antibody binding preference (for this PND peptide and these antibodies) is then discussed.

EXPERIMENTAL PROCEDURES
Peptide Synthesis-A N-terminal cysteinylated form of peptide RK known as CRK, Cys Ϫ1 -Arg 1 -Ile 2 -Gln 3 -Arg 4 -Gly 5 -Pro 6 -Gly 7 -Arg 8 -Ala 9 -Phe 10 -Val 11 -Thr 12 -Ile 13 -Gly 14 -Lys 15 , was synthesized and studied. The cysteine residue was required for conjugation of RK peptide to BPTI protein via a heterobifunctional chemical linker. This 16-residue peptide was synthesized using a Rainin Automated PS-3 Peptide Synthesizer and Fmoc chemistry. The crude peptide was purified using a Rainin HPXL HPLC System equipped with a Rainin Dynamax-300A reverse phase column with acetonitrile as the elution solvent. The peptide composition was verified by amino acid analysis and its molecular weight by FAB mass spectroscopy (Multiple Peptide Systems, San Diego, California). The peptide sequence and its purity (Ͼ95%) were evaluated by two-dimensional NMR techniques.
BPTI Modification and Peptide Coupling-The modification of BPTI and its coupling to CRK peptide was accomplished according to the method developed by Ebina et al. (34) with modifications. BPTI was modified using OMIU and then covalently linked to the chemical linker, SPDP, before peptide coupling. The modification of BPTI proceeds by conversion of its lysine residues to homoarginines. As a result, the N-terminal amino group remains as the only free amino group available for SPDP coupling. The cysteinyl side chain of the peptide is covalently attached to this linker. The protein was modified using o-methylisourea and then purified using a Amersham Pharmacia Biotech LKB FPLC system and a Mono-S ion exchange column. A NaCl salt gradient in a 20 mM glycine, pH 10.5, buffer was used to purify the modified protein. The main protein fraction, which eluted at NaCl concentrations greater than 0.54 M, was collected. This fraction was then dialyzed extensively against deionized, distilled water and subsequently lyophilized. The molecular weight of the OMIU-modified BPTI (OMIU-BPTI) was determined via matrix-assisted laser desorption/ionization-time of flight mass spectroscopy to be 6681 (versus 6513 for native BPTI), the expected mass for correctly modified BPTI. Amino acid analysis also conducted upon this protein further verified that conversion of the lysine side chains had been achieved. CRK peptide was then coupled via its terminal cysteine side chain to OMIU-BPTI using the cross-linker, SPDP. A disulfide bond between the peptide cysteine sulfhydryl group and linking reagent (shown in brackets below) is formed to produce the final protein-peptide conjugate: BPTI-NH-{CO-CH 2 -CH 2 -S}-S-Cys-RK Peptide. "BPTI-CRK" is the abbreviation used to denote CRK peptide conjugated to OMIU-BPTI. The conjugate was then purified using an FPLC and a Mono-S column. The conjugate was eluted using a 0 -100% 1 M NaCl gradient in 100 mM sodium phosphate, pH 7.5. The conjugate eluted at ϳ0.95 M NaCl with this gradient and the fraction collected at this salt concentration was then dialyzed against 0.1 M NaCl followed by de-ionized, distilled water via ultrafiltration and an Amicon YM-3 membrane.
NMR Sample Preparation-NMR samples of CRK peptide, OMIU-BPTI, and BPTI-CRK peptide conjugate were prepared in 90% H 2 O, 10% D 2 O solutions at pH 4.1 with typical sample volumes of 0.5 ml. The various sample concentrations were 5.7 mM(CRK), 6.0 mM (modified BPTI), and 5.9 mM (BPTI-CRK). The pH of all NMR samples (uncorrected for deuterium isotope effects) was adjusted to 4.1 using DCl and NaOD solutions and an Orion520A pH meter. All proton resonances were ultimately referenced against DSS. To maintain the uncoupled cysteinylated peptide in its reduced state, dithiothreitol-d 6 was added to these samples using a molar ratio of dithiothreitol:peptide ϭ 50:1.
NMR Spectroscopy-A Bruker DMX 500MHz spectrometer was used to acquire the NMR data presented. Standard NMR experiments and pulse sequences such as DQF-COSY (35,36), NOESY (37), TOCSY (38), and double quantum experiments (39,40) were used for assignment purposes. The water resonance was suppressed via the use of low level rf pulses applied at the beginning of the pulse sequence (usually 1.5 s long) and during the mixing time of a NOESY sequence. The sweep widths used were 5000 -8012 Hz. The number of points collected was 8192 or 4096 during t 2 and 512 or 1024 points during t 1 . For each t 1 point, 32 scans were acquired for all the NMR experiments. All NMR probe temperatures were calibrated using neat methanol (41).
The NMR data were processed using Felix 2.30 (Biosym Technologies, Inc.) software run either on a Silicon Graphics INDIGO R4000 or a Sun SPARCstation5 computer. Typical processed two-dimensional data matrices were 4 K by either 1 or 2 K. A /2 phase-shifted sinebell window function was applied during F1 and F2 processing. Prior to F1 transformation, the first interferogram was multiplied by 0.5 to suppress t 1 ridges (42). The intensities of NOESY cross-peaks were measured by calculating the volumes of the cross-peaks as the integral of all the data point intensities within the cross-peak footprints. For the NOE intensity measurement of free and coupled peptides, the relative intensities of these NOEs were calibrated against the d ␣N (i, iϩ1) NOE observed between the F10 H␣ and V11 HN protons of each peptide.
Fab Preparation-Antibodies 5023A and 5025A were cleaved using papain and purified following standard procedures (43). All buffers were degassed before use and most steps in the procedure were conducted under N 2 gas. The enzyme and antibody solutions were mixed in a 1:100 papain:antibody ratio. The cleavage proceeded at 310 K for 12 h before the addition of iodoacetamide to a final concentration of 40 mM. This mixture was then incubated at 298 K for 1 h. The Fab was then dialyzed against a phosphate-buffered saline, pH 7.0, buffer using an Amicon and a YM30 membrane. The Fab fragment was purified with a FPLC system equipped with a Pharmacia 5-ml pre-packed protein A column using a pH 7.0 phosphate-buffered saline wash buffer and a 50 mM citrate, pH 3.0, elution buffer.
SPR Experiments-Binding kinetics were determined by SPR using a BIAcore 1000 TM biosensor system (Biacore Inc., Piscataway, NJ) (44). The BPTI-peptide conjugate and gp120 (strain LAV, obtained from MicroGeneSys, Inc. via the NIH AIDS Research and Reference Reagent Program) were immobilized on research grade CM5 sensor chips at concentrations of 5 mg/ml in 10 mM sodium acetate, pH 4.5, for the conjugate and 10 mM sodium acetate, pH 6, for gp120 using the amine coupling kit supplied by the manufacturer. Approximately 150 resonance units of each conjugate and 2500 resonance units of gp120 were immobilized; one RU corresponds to an immobilized protein concentration of ϳ1 pg/mm 2 (45). Unreacted moieties on the surface were blocked with ethanolamine. The N-terminal cysteine of CRK was used to immobilize the peptide onto research grade CM5 sensor chips using a thiol-exchange coupling procedure. The carboxylated dextran surfaces of the sensor chips were first activated with N-hydroxysuccinimide and N-ethyl-NЈ-(dimethylaminopropyl)carbodiimide. Reactive disulfides were then introduced by reaction with 2-(2-pyridinyldithio)ethaneamine in 100 mM borate buffer, pH 8.5. The N-terminal cysteinylated peptide was then introduced at concentrations of 5 and 0.5 mg/ml, respectively, in 10 mM acetate buffer, pH 4.5. Finally, unreacted moieties on the surface were blocked with cysteine. All measurements of 5023A and 5025A Fab binding to the conjugate, gp120, and peptide surfaces were carried out in HEPES-buffered saline which contained 10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA. Analyses were performed at 298 K and flow rates of 30 -50 ml/min. All surfaces were regenerated with 100 mM H 3 PO 4 .
SPR Data Analysis-Association and dissociation rate constants were calculated by numerical integration and global fitting to a 1:1 interaction model using BIAevaluation 3.0 software (Biacore, Inc.) and the equation: is the response at time t, R max is the maximum response, C is the concentration of analyte in solution, k a is the association rate constant, k d is the dissociation rate constant, and RU (0) ϭ 0.

Proton Resonance
Assignments-Proton chemical shift assignments for uncoupled and BPTI-coupled CRK peptide (Table I) were made on the basis of DQF-COSY, TOCSY, and NOESY data obtained from these samples according to standard assignment procedures (46). The spin systems were established from DQF-COSY and TOCSY data, while sequential resonance assignments were determined from NOESY data collected on these samples. All resonance assignments reported here for the various peptide and BPTI forms were obtained at 298 K and pH 4.1.
In the uncoupled and coupled CRK peptide spectra (Table I), a major, as well as minor, set of resonances was observed for residues Gly 5 to Arg 8 . Based upon the d ␣ ␣ ␣ (i, iϩ1) NOEs observed between the H␣ protons of the minor form of Gly 5 and Pro 6 that were measured from D 2 O samples of free CRK and peptide (data not shown), the minor resonances were attributed to the cis form of these peptides arising from cis-trans isomerization about the Gly 5 -Pro 6 peptide bond. The amount of cis conformer present in both peptide forms was estimated to be close to 10%.
BPTI Modification and Peptide Coupling-Nearly complete proton resonance assignments for native, 2 OMIU-modified, and peptide-conjugated BPTI were obtained in order to determine whether chemical modification and peptide coupling significantly affected BPTI. This was accomplished via comparison of the native BPTI versus OMIU-BPTI HN and H␣ resonance frequencies. Very few resonance frequency changes were observed for most BPTI residues regardless of BPTI form and most of these were less than 0.02 ppm. The largest changes (observed after linkage of SPDP to OMIU-modified BPTI) consisted of large frequency shifts involving the following protein resonances: the HN and H␣ of Ala 58   converted to an amide after peptide coupling. Uncoupled Versus Coupled Peptide NOE Studies-The uncoupled peptide gave rise to medium-strong d ␣N (i, iϩ1) NOEs involving residues Arg 1 through Gly 14 (Fig. 1A). A weak d ␣N (i, iϩ1) NOE was also observed between Arg 1 and Ile 2 . At the same time, a few medium to weak d NN (i, iϩ1) NOEs were detected between residues Gly 7 -Thr 12 . Other sequential NOEs observed included a d ␤N (i, iϩ1) NOE between Ala 9 and Phe 10 . In addition, strong d ␣␦ (i, iϩ1) NOEs between both Gly 5 H␣ protons and the Pro 6 H␦ proton were measured. Intraresidue d N␣ (i, i) NOEs were observed for residues Gly 5 , Gly 7 , Val 11 , Thr 12 , and Gly 14 . This type of NOE was also observed for Arg 8 . The intraresidue d N␣ (i, i) NOEs of Arg 4 and Ala 9 were overlapped with sequential d ␣N (i, iϩ1) NOEs between Gln 3 -Arg 4 and Arg 8 -Ala 9 , respectively.
As observed for the uncoupled peptide, most of the backbone protons of the coupled peptides gave rise to strong d ␣N (i, iϩ1) NOEs (Fig. 1B). For coupled CRK peptide, d NN (i, iϩ1) NOEs were measured between sequential residues beginning with Ile 2 -Thr 12 (except Gln 3 -Arg 4 and Ala 9 -Phe 10 ). The strongest d NN (i, iϩ1) NOEs involved residues Gly 7 -Ala 9 . Furthermore, a weak Pro 6 -Arg 8 d ␣N (i, iϩ2) NOE was observed ( Fig. 2A). For residue Pro 6 , strong d ␣␦ (i, iϩ1) NOEs between Gly 5 and Pro 6 were observed. Additionally, a d N␦ (i, iϩ1) NOE between the Gly 5 HN and Pro 6 H␦ protons was observed for coupled CRK. Another weak, but significant d ␦N (i, iϩ1) NOE between the Pro 6 H␦ and Gly 7 HN protons was also detected (Fig. 2B). Other sequential NOEs observed included d ␤N (i, iϩ1) NOEs between residues Ile 2 -Gln 3 , Ala 9 -Phe 10 , and Val 11 -Thr 12 . For the conjugated peptide, strong to medium intraresidue d N␣ (iϩ1) NOEs were measured, except for residue Phe 10 , for which a weak NOE of this type was measured instead. This type of NOE was not observed for Cys Ϫ1 and Arg 1 . Intraresidue d N␣ (iϩ1) NOEs of Arg 4 and Ala 9 were overlapped with d ␣N (i, iϩ1) NOEs of Gln 3 -Arg 4 and Arg 8 -Ala 9 , respectively. The corresponding Ile 13 NOE was overlapped with an intramolecular BPTI NOE.
The ␤Ϫturn NOEs observed for conjugated CRK were absent in the corresponding free peptide NOESY data, regardless of the mixing time (100 -600 ms) used. To verify whether this discrepancy was a consequence of the peptide correlation time, free peptide ROESY data 2 were also collected (data not shown). The overall pattern of peptide resonances and the relative intensities of the free CRK cross-peaks measured were similar in the ROESY versus the NOESY data. Regardless of ROESY mixing time (50 -300 ms, data not shown) used, GPGR ␤Ϫturn cross-peaks were not observed at 298 K and 500 MHz in free CRK ROESY data. Since the NOESY and ROESY experiments were equally sensitive as far as detection of peptide crossrelaxation peaks, the NOESY experiment was employed since it allowed for better observation of BPTI resonances in the BPTI-CRK conjugate.
Antibody Binding Kinetics and Affinity Measurements-SPR was used to determine the kinetics and affinities of 5023A and 5025A binding to the following antigens: uncoupled and coupled CRK peptide and native gp120 protein. The SPR studies employed an antigen surface that was produced by immobilizing the antigen onto a dextran surface. The Fab (5023A or 5025A) was then flowed over this surface. Good fits to a 1:1 interaction model were obtained for most of the 5023A SPR sensorgram data. The overall quality of the fit is reflected in the relative errors of the affinity (K Aff ) constants derived from the sensorgram data. Table II summarizes the 5023A and 5025A K Aff values, association (k a ) and dissociation (k d ) rates measured for all the antigens. By SPR, the affinities of both antibodies for RK and CRK were measured to be the same, within experimental error (data not shown). The 5023A and 5025A affinities for RK peptide (data not shown) were similar to those published earlier and determined by modified enzymelinked immunosorbent assay (47). It was determined from microcalorimetry experiments that 5023A and 5025A do not bind appreciably to BPTI (data not shown). Fig. 3 provides an example of the 5023A and 5025A sensorgram data used to obtain the values listed in Table II. The data for 5023A Fab binding to CRK and gp120 fit well to a 1:1 interaction model but the data for 5023A Fab binding to BPTI-CRK gave somewhat inferior fits and larger errors (Table  II). In general, the affinities for the various antigens were relatively high (Ͼ10 8 M Ϫ1 ) and not dramatically different. The relative error associated with the measured CRK and gp120 affinities were both relatively low (ϳ5% or less) indicating that the 7-fold higher affinity for CRK relative to gp120 is significant. The 5023A/BPTI-CRK affinity had the highest (ϳ10%) relative error among all the measured affinities for both antibodies (see Table II). In terms of binding kinetics, the association rates for CRK and BPTI-CRK were all similar whereas the corresponding dissociation rates for these complexes differed considerably. The BPTI-CRK dissociation rate was nearly twice that of CRK and this accounts for the corresponding decreased BPTI-CRK antibody affinity. Despite the difference in gp120 versus CRK association rates, their dissociation rates were

FIG. 1. Backbone NOE connectivities observed for the free (trans) CRK (A) and coupled (trans) CRK (B).
These charts were based upon NOESY (500-ms mixing time) data recorded from these peptides at 500 MHz, 298 K, and pH 4.1. The lines drawn correspond to NOEs observed between the indicated protons and residues. Relative intensities of these NOEs are represented by relative line thickness. The d ␣␦ (i, iϩ1) NOE between Gly 5 H␣ and Pro 6 H␦ protons represent the average of the two d ␣N (i, i) and d ␣␦ (i, iϩ1) NOEs between the two nondegenerate Gly 5 H␣ protons and the Pro 6 H␦ protons. The d N␣ (i, i) of Gly 5 is the average of the two d N␣ (i, i) NOEs between the two nondegenerate Gly 5 H␣ protons and its HN proton. "*" indicates resonance overlap.
nearly the same. In either case, both of these antigens dissociated from the 5023A at a slower rate than did BPTI-CRK.
The Fab binding kinetics and affinity data obtained were more uniform in the case of 5025A with the binding to all three antigens fitting well to a 1:1 interaction model with low relative errors. The typical 5025A affinity (K Aff ) was approximately 10 6 M Ϫ1 and this was about 100-fold lower than the average 5023A affinity. The preferred order of 5025A binding, BPTI-CRK Ͼ CRK Ͼ gp120, should be accurate in view of the good fitting to the interaction model in each instance. Relative to gp120, the k a and k d rates for CRK peptide (regardless of conjugation state) binding were both somewhat faster. DISCUSSION

BPTI Modification and Peptide
Coupling-The largest effects observed upon BPTI due to peptide conjugation were restricted to the C-and N-terminal regions of BPTI. These effects were seen only after BPTI linkage to SPDP and consisted of substantial changes in the Ala 58 and Arg 1 backbone resonance frequencies. At the pH (4.1) of these studies, a salt bridge exists between the ␣-amino group of Arg 1 and the Cterminal carboxylate group of residue Ala 58 in native BPTI (48,49). Since the ␣-amino (or N) terminus of BPTI becomes an amide group after BPTI reaction with SPDP, a relatively large (Ͼ0.2 ppm) shift of the BPTI Arg 1 H␣ resonance occurred after SPDP coupling. Substantial shifts of the BPTI Ala 58 H␣ and HN resonances were also observed upon SPDP coupling and peptide linkage. The simultaneous occurrence of frequency changes involving the backbone Ala 58 and Arg 1 resonances is consistent with disruption of the native BPTI Arg 1 -Ala 58 BPTI salt bridge due to conjugation of peptide to BPTI.
Since only minor NMR changes were observed for the remaining backbone resonances of BPTI after conjugation, the backbone of BPTI appears to be mostly unperturbed by such conjugation. This, along with the lack of significant CRK peptide resonance changes (regardless of peptide coupling), suggest that CRK and BPTI do not interact with each other significantly in the conjugate. This is supported by NOE build-up data for the free and conjugated forms of the peptide and BPTI (data not shown) which indicate that the overall correlation times ( c ) of BPTI and CRK are not significantly affected by conjugation. The backbone CRK NOE maximum remains the same (regardless of conjugation to protein) and occurs at a much longer mixing time (400 -500 ms, data not shown) compared with BPTI for which the NOE maximum remains between 100 and 150 ms (regardless of peptide coupling). However, relaxation studies of CRK peptide indicate that attachment of the peptide does affect CRK dynamics to some extent. 3 Structural Tendencies of Uncoupled Versus BPTI-Coupled CRK Peptide-By NMR, free CRK peptide was determined to be mostly unstructured in solution. This is based upon the simultaneous observation of numerous, relatively strong sequential d ␣N (i, iϩ1) NOEs and a few relatively weak d NN (i, iϩ1) NOEs throughout the peptide (50). In addition, the significant absence of medium-to long-range NOEs indicates that CRK adopts very little folded structure and that it undergoes extensive conformational averaging in solution. Other NMR evidence in support of such averaging was provided by the averaged J N␣ constants 2 (5.7-7.7 Hz, data not shown) and H␦ and Gly 7 HN protons. "G 5 N-P 6 ␦" corresponds to the d N␦ (i, iϩ1) NOE between the Gly 5 HN and Pro 6 H␦ protons.
FIG. 2. NOESY data recorded from free and coupled forms of CRK peptide at 500 MHz using a 500-ms mixing time. A, upper panel corresponds to the f2 row at the Pro 6 H␣ proton frequency in the NOESY spectrum of free CRK; lower panel is the analogous row in the NOESY data obtained from coupled CRK. The NOEs observed between the Pro 6 H␣ and Gly 7 HN and the Pro 6 H␣ to Arg 8 HN proton of the coupled peptide are indicated. B, fingerprint region of the NOESY spectrum. "P 6 ␦ϪG 7 N" corresponds to the d ␦N (i, iϩ1) NOE between Pro 6 relatively high values for the backbone amide proton temperature coefficients 2 (Ϫ5.3 to Ϫ9.1 ppb/K, data not shown). The strong intensities of the d ␣N (i, iϩ1) NOEs are indicative of an extended peptide backbone. The peptide also exhibits a slight tendency to adopt some non-extended conformation involving residues Gly 7 -Thr 12 , based upon the weak d NN (i, iϩ1) NOEs observed for these residues.
The BPTI-coupled peptide NOEs also consisted primarily of strong backbone d ␣N (i, iϩ1) NOEs and less frequently, weaker intensity d NN (i, iϩ1) NOEs. In cases where both NOE types were observed simultaneously, the relative intensity of the sequential d ␣N (i, iϩ1) NOE was always much higher. The coupled peptide backbone was also mostly extended and the peptide was still conformationally averaged after BPTI coupling. Since d NN (i, iϩ1) NOEs were observed for residues Ile 2 to Thr 12 in coupled CRK, the middle to N-terminal portion of the peptide backbone was less extended relative to the remainder of the peptide. An alternative explanation for the higher intensities of these particular d NN (i, iϩ1) NOEs may be that these residues are more motionally restricted due to their proximity to the protein attachment site.
In terms of NOEs specific to non-extended backbone structure, the coupled peptide Gly 7 -Ala 9 d NN (i, iϩ1) NOEs were stronger relative to other d NN (i, iϩ1) NOEs and these residues therefore adopt non-extended conformations more readily. However, two new, but weak NOEs were observed for the coupled peptide. One of these corresponds to a d ␣N (i, iϩ2) NOE between the Pro 6 H␣ and Arg 8 HN protons, and it reflects a small ␤Ϫturn tendency in the GPGR region. The second is a d ␦N (i, iϩ1) NOE between the Pro 6 H␦ and Gly 7 HN protons and it is further consistent with a type I ␤-turn in this region. However, the concurrent existence of a type II ␤-turn cannot be ruled out on the basis of these data alone since a strong d ␣N (i, iϩ1) NOE between the Pro 6 H␣ and Gly 7 HN protons was also observed. This latter NOE is characteristic of either a type II ␤-turn comprised of residues Gly 5 through Arg 8 or an extended backbone involving residues Pro 6 and Gly 7 . Based on the relatively low intensity of this d ␣N (i, iϩ2) NOE, the ␤-turn tendency is therefore significant but weak.
The d ␣N (i, iϩ2) and d ␦N (i, iϩ1) cross-peaks were unobservable in NOESY and ROESY 2 data recorded from the free peptide. While it is possible that the GPGR turn tendency exists in the unconjugated peptide but was undetectable via NOESY due to peptide correlation time, these ␤-turn cross-peaks should still have been detected via the ROESY experiments that were also performed. Therefore, this structural tendency probably does not exist in the free peptide, or if it does, it is too weak for detection.
In contrast to an earlier study of a nonapeptide conjugated to BPTI (26), the conjugation of BPTI to CRK did not significantly affect the structural tendencies of CRK. This may simply reflect differences between these two peptides in terms of their inherent structural tendencies.
The NMR results reported here for uncoupled (or free) CRK peptide are consistent with other NMR investigations conducted by numerous groups upon a variety of V3 loop-derived peptides (8 -21). The peptides studied varied somewhat in length (12-24 residues) and sequence but they all contained the highly conserved V3 loop GPGR residues. Typically, the overall tendency of these peptides to adopt a ␤-turn (of either type I or II) conformation in the GPGR region was relatively low. The peptides studied that were similar in length to CRK were shown to be primarily extended and mostly unfolded in solution. The relative intensities of the GPGR ␤-turn NOEs observed for other V3 loop peptides (similar to CRK in length) were comparable to those of coupled CRK peptide (see above). Therefore, the relative populations of ␤-turn structure adopted by those peptides versus coupled CRK in aqueous (without added trifluoroethanol) solution are about the same.
Antigen Structure versus Antibody Binding-The binding data indicate that 5025A antibody binds to both forms of CRK slightly better than gp120. For 5023A, the observed affinity differences are somewhat larger (Ͻ7-fold) and free CRK is preferred equally over the conjugated peptide and gp120 antigens. However, since the 5023A/BPTI-CRK sensorgram data fit least well to the interaction model, some caution must be exercised in interpretation of 5023A binding differences involving this antigen and others such as CRK, for example. The lower quality of the 5023A/BPTI-CRK SPR data is most likely attributable to effects upon 5023A binding due to heterogeneous  coupling of BPTI-CRK to the binding surface.
Since all the antigens (except CRK) were coupled via their amino groups, they were heterogeneously coupled to this surface. CRK was attached via its cysteine side chain, and was homogeneously coupled instead. Heterogeneous antigen coupling effects upon antibody binding are more significant for 5023A binding to the peptide conjugate than it is for gp120, since the latter has many more non-epitope amino groups available for surface immobilization.
Non-homogeneous antigen coupling to the surface can lead to complex binding behavior if coupling results in a mixture of fully and partially active antigen. This in turn will lead to complex Fab binding behavior and poorer fits of the obtained SPR data to simple interaction models. Such surface coupling heterogeneity is anticipated to yield SPR data with a faster than expected dissociation rate and a higher relative error for the value of the antibody affinity. Both of these phenomena were observed in the case of the 5023A/BPTI-CRK SPR data and the corresponding affinity. Therefore, the 5023A 7-fold binding difference between the different forms of CRK is probably not meaningful.
In contrast to 5023A, all the 5025A-conjugate sensorgram data were fit well to a 1:1 interaction model. Correspondingly, the relative errors obtained for the 5025A K Aff values were all lower compared with those of 5023A. This discrepancy can be rationalized on the basis of differences in the 5023A and 5025A antigenic epitopes (47,51). The 5023A RK epitope consists of residues Arg 4 , Pro 6 , Gly 7 , Arg 8 , Ala 9 , Phe 10 , Thr 12 , and Gly 14 , whereas the 5025A epitope is smaller and involves only residues Pro 6 , Arg 8 , Ala 9 , and Phe 10 . Since all the 5025A epitope residues are distant from both peptide coupling sites, heterogeneous coupling effects upon 5025A binding to the conjugate were probably much less significant. For 5023A, however, residue Gly 14 is a 5023A epitope residue that is adjacent to Lys 15 ; hence 5023A binding to BPTI-CRK peptide could be hindered due to surface coupling of this peptide via its Lys 15 side chain.
The differences in antibody binding to the various antigens (discussed above and summarized in Table II) were all meaningful, with the possible exception of the 5023A binding to CRK-BPTI data. However, all these binding differences were Ͻ10-fold and corresponded to Յ1 kilocalorie/mol affinity changes. Hence, antibodies 5023A and 5025A both exhibited no strong binding preference for any of the antigens, CRK, BPTI-CRK, and gp120.
To fully determine the structural basis for the antigenic binding differences observed for the 5023A/5025A antibodies, a detailed structural comparison between the intact gp120 V3 loop versus the 5023A/5025A antibody-bound state of the V3 loop (i.e. RK) peptide is really required. Such structural information regarding the V3 loop of gp120 (22), as well as the corresponding 5023A/5025A antibody-bound structures of gp120 and RK, are still unavailable, however. Nonetheless, general insight into this problem can still be derived from the structure and affinity data presented here. Based upon the fact that 5023A and 5025A are both cross-reactive (i.e. they bind to both the peptide and protein antigens), and that RK peptide is relatively unstructured in solution, there are at least three general schemes of anti-peptide antibody binding to consider as shown below (52). It is assumed in all cases that a substantial binding difference corresponds to a 10-fold or higher affinity change and that the antibody conformation remains the same, regardless of antigen binding. (a) If the anti-peptide antibody binds substantially better to native protein, the native protein structure is strongly preferred by this antibody. (b) If the antipeptide antibody binds substantially better to the peptide than the native protein, unfolding of the protein is probably in-volved. The extent of such unfolding will be reflected by the extent to which the protein affinity is decreased relative to that of the peptide. (c) If the anti-peptide antibody binds the peptide and protein with nearly equal affinity, conformational differences between the protein and peptide are most likely small.
Typically, anti-peptide antibodies bind preferentially (and sometimes exclusively), to the "unfolded" protein fragment or peptide versus the folded protein (53)(54)(55)(56)(57). 5023A and 5025A are both anti-peptide antibodies raised against a protein-conjugated form of RK peptide. Both antibodies behaved "typically" in that they exhibited slightly higher affinities for the less structured peptide versus the native protein. This preference, however, was slight since the largest measured protein-peptide affinity difference corresponded to an energetic change of only about 1 kilocalorie/mol. Therefore, both antibodies exhibited essentially the same affinity for the protein and the peptide, regardless of conjugation. From the 5023A and 5025A affinity data, these antibodies are therefore examples of c above.
One of the simplest interpretations of the 5023A and 5025A binding data is that structural differences between CRK peptide and the corresponding V3 loop residue sequence in gp120 protein are probably small. Therefore, only small conformational changes of the protein may be required for its binding to 5023A and 5025A. CRK peptide was shown by NMR to adopt very little persistent structure. Hence the relevant residues in gp120 are also likely to be correspondingly unstructured and conformationally heterogeneous. This is entirely consistent with the fact that these residues are located in a loop region of gp120 protein.
Further evidence that the V3 loop is conformationally heterogeneous is provided by structural studies of various antibody-V3 loop peptide complexes (58 -62). Structural comparison of two different V3 loop peptide-antibody complexes reveals that the structures adopted by the antibody-bound peptides in the two complexes were quite dissimilar and antibody-dependent (58,60). This is despite the fact that both antibodies (0.5␤ and 59.1) neutralize the same strain of HIV-1 (IIIB) and therefore recognize and bind the same gp120 protein. Based upon these data, the gp120 IIIB V3 loop (from which the CRK sequence is derived) is probably conformationally heterogeneous.
From further structural comparison of the antibody-bound versus unbound V3 loop peptides, it appears that the structure of the peptide was typically quite different in these two states (10, 58 -62). This observation is also consistent with the observed lack of significant folded structure among the V3 loop peptides studied, as well as the important influence of the antibody upon the final antibody-bound structure adopted by these peptides.
For antibodies 5023A/5025A, CRK antigenicity does not appear to depend upon ␤-turn structure adopted in the GPGR region. This is based upon: 1) the relatively high affinity binding of these antibodies to CRK which is relatively unstructured, and 2) the fact that the small tendency of this peptide (in its conjugated state) did not significantly affect 5023A/5025A binding to the peptide. This is also consistent with the fact that the full epitopes recognized by these antibodies include most, but not all of the residues in the GPGR sequence (47,51) and hence adoption of any particular structure in the GPGR region may be of less consequence to antibody binding. Also, since CRK lacks significant folded structure, the most likely scheme of peptide binding by antibodies 5023A and 5025A must involve an induced fit of this peptide to the binding sites of both antibodies.
In terms of HIV-1 virus neutralization, the binding of neutralizing antibodies to HIV-1 virus appears to somehow interfere with specific, but still unknown HIV-1 V3 loop binding events that are required for infection (63). While the 5023A and 5025A antibodies are not necessarily representative of all HIV-1 neutralizing antibodies, it appears that their binding to HIV-1 is independent of the particular, folded structure adopted by the V3 loop GPGR residues. If a conserved structure adopted by these conserved residues is not crucial for 5023A/ 5025A neutralization of HIV-1, other possible functions for these residues should be considered. One proposed possibility is that this V3 region is directly involved in the binding and interaction between HIV-1 and co-receptor protein(s) (64). 5023A/5025A binding to this region may prohibit important co-receptor interactions involving these conserved gp120 residues. Another potential function of this conserved residue sequence may be that it serves as a recognition site for proteolysis of the V3 loop; specific cleavage of the V3 loop has been postulated to precede the cell membrane fusion events required for virus infection (61,(65)(66)(67). Flexibility of the V3 loop, especially involving the GPGR residues, would also be beneficial in this situation (66). For this latter possibility, 5023A/5025A binding to a particular region of the V3 loop could serve to prohibit access to this critical cleavage site (68).


extfieldsofstudy: Biology Medicine

text:
The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli.

In Escherichia coli a subset of periplasmic proteins is exported through the Tat pathway to which substrates are directed by an NH(2)-terminal signal peptide containing a consensus SRRXFLK "twin arginine" motif. The importance of the individual amino acids of the consensus motif for in vivo Tat transport has been assessed by site-directed mutagenesis of the signal peptide of the Tat substrate pre-SufI. Although the invariant arginine residues are crucial for efficient export, we find that slow transport of SufI is still possible if a single arginine is conservatively substituted by a lysine residue. Thus, in at least one signal peptide context there is no absolute dependence of Tat transport on the arginine pair. The consensus phenylalanine residue was found to be a critical determinant for efficient export but could be functionally substituted by leucine, another amino acid with a highly hydrophobic side chain. Unexpectedly, the consensus lysine residue was found to retard Tat transport. These observations and others suggest that the sequence conservation of the Tat consensus motif is a reflection of the functional importance of the consensus residues. Tat signal peptides characteristically have positively charged carboxyl-terminal regions. However, changing the sign of this charge does not affect export of SufI.

In Escherichia coli a subset of periplasmic proteins is exported through the Tat pathway to which substrates are directed by an NH 2 -terminal signal peptide containing a consensus SRRXFLK "twin arginine" motif. The importance of the individual amino acids of the consensus motif for in vivo Tat transport has been assessed by site-directed mutagenesis of the signal peptide of the Tat substrate pre-SufI. Although the invariant arginine residues are crucial for efficient export, we find that slow transport of SufI is still possible if a single arginine is conservatively substituted by a lysine residue. Thus, in at least one signal peptide context there is no absolute dependence of Tat transport on the arginine pair. The consensus phenylalanine residue was found to be a critical determinant for efficient export but could be functionally substituted by leucine, another amino acid with a highly hydrophobic side chain. Unexpectedly, the consensus lysine residue was found to retard Tat transport. These observations and others suggest that the sequence conservation of the Tat consensus motif is a reflection of the functional importance of the consensus residues. Tat signal peptides characteristically have positively charged carboxyl-terminal regions. However, changing the sign of this charge does not affect export of SufI.
Proteins that are exported to the periplasm or periplasmic face of the bacterial cytoplasmic membrane are usually synthesized with an NH 2 -terminal signal peptide that mediates targeting to the export apparatus. After translocation of the precursor protein to the periplasm the signal peptides are normally removed by the enzyme signal peptidase. The majority of periplasmic proteins are exported via the Sec apparatus. A major feature of the Sec apparatus is that proteins are translocated in an extended conformation and are often bound by SecB or other cytoplasmic chaperones to prevent folding prior to export (1)(2)(3). Sec pathway signal peptides are typically 18 -26 amino acids in length. Although conserved amino acid sequences are absent, Sec signal peptides possess a common tripartite structural organization comprising a positively charged amino-terminal (n-) 1

region, a central hydrophobic (h-)
region with a high propensity for ␣-helix formation, and a carboxyl-terminal (c-) region that carries the cleavage site for the signal peptidase (4 -6).
It has recently become clear that bacteria possess a second general protein export pathway that is quite distinct from the Sec apparatus (7)(8)(9)(10)(11)(12). This Sec-independent pathway has been termed the Tat (for twin arginine translocation) system (12) because precursors are targeted to the pathway by signal peptides that, while preserving the tripartite structural organization of Sec signal peptides, bear a characteristic n-region sequence motif that includes two consecutive and invariant arginine residues. Studies in Escherichia coli suggest that the Tat system is minimally comprised of the four integral membrane proteins TatA, TatB (also termed MttA2; GenBank accession no. AF067848), TatC, and TatE (11)(12)(13)(14). Remarkably and uniquely this translocase functions to transport folded proteins across the cytoplasmic membrane, a feat that must be achieved without rendering the membrane freely permeable to protons and other ions.
The thylakoid membrane of plant chloroplasts contains protein transport systems homologous to both the Sec and Tat pathways of bacteria (15). Targeting to the Tat-analogous thylakoid ⌬pH-dependent transport pathway is, like its bacterial counterpart, mediated by signal peptides ("transfer peptides" or "lumen-targeting domains") with paired arginine residues (16), and indeed bacterial Tat signal peptides have been shown to direct efficient thylakoid import specifically by the ⌬pH-dependent system (17)(18)(19). In both bacteria (6,20) and chloroplasts (21,22) the signal peptide alone is capable of mediating mutually exclusive sorting of precursor proteins between the Tat and Sec pathways. Thus, even though they conform to the tripartite structure of Sec signal peptides, Tat signal peptides avoid functional interactions with the Sec apparatus. Recent studies in bacteria suggest that the low h-region hydrophobicity of Tat signal peptides relative to that of Sec signal peptides is the major structural feature precluding targeting of Tat substrates to the Sec pathway (6), and the same mechanism may apply in chloroplasts (23). In addition, basic residues commonly found in the c-region of bacterial and thylakoid Tat signal peptides, but characteristically absent from the c-region of Sec signal peptides, have been shown in thylakoids to operate as a "Sec-avoidance" determinant (24). A similar function is likely for these residues in bacterial Tat signal peptides given the longstanding observations that introduction of positive charges in the vicinity of the leader peptidase site slows transport of Sec-targeted precursors (e.g. Refs. 6 and 25).
Targeting to the Tat apparatus in chloroplasts exhibits an absolute dependence on the invariant twin arginine pair within the signal peptide (16,23). Studies with bacterial Tat signal peptides suggest that the twin arginine residues are also important in targeting to the bacterial Tat pathway (6, 26 -29). However, a major difference between bacterial and plant Tat signal peptides is that the bacterial signal peptides contain additional conserved amino acids surrounding the paired arginines. A consensus for this extended twin arginine motif has been defined as SRRXFLK where the arginine residues are invariant, and the frequency of occurrence of the other amino acids exceeds 50% (7). The observation of an extended n-region motif immediately raises the question of the role and importance of the non-arginine consensus residues in the bacterial Tat transport process. A further obvious question is whether these additional potential targeting determinants reduce the importance of the arginine pair that is essential for thylakoid Tat transport. In an attempt to address these questions we have undertaken a systematic site-directed mutagenesis study of the extended bacterial Tat consensus motif and assessed the effect of these signal peptide alterations on protein export. In addition, we have investigated the importance of the c-region charge in targeting to the bacterial Tat apparatus.
During all genetic manipulations E. coli strains were grown aerobically in Luria-Bertani medium (35). Concentrations of antibiotics were as described previously (12).
Plasmid Construction-Plasmids for the in vivo synthesis of pre-SufI and pre-YacK were constructed as follows. A 1,430-base pair polymerase chain reaction fragment covering the region from 18 nucleotides upstream of the start codon of sufI, including the Shine-Dalgarno sequence, to the stop codon was amplified using the primers 5Ј-GCGC-GAATTCGTTTTACATGGAGCAAATATG-3Ј and 5Ј-GC GCTCTAGAT-TACGGTACCGGATTGACC-3Ј with MC4100 chromosomal DNA as the template. The product was digested with EcoRI and XbaI and cloned into the polylinker of pT7.5 (36) to give plasmid pNR14, which was used as the template for all subsequent mutations in the signal peptide coding portion of sufI. A 1,464-base pair DNA fragment covering the region from 16 nucleotides upstream of the start codon of yacK to the stop codon was amplified from MC4100 chromosomal DNA using the primers 5Ј-GCGCGAATTCGAATAAGGAAATAACTATG-3Ј and 5Ј-GCGCTCTAGATTATACCGTAAACCCTAAC-3Ј. The polymerase chain reaction product was digested with EcoRI and XbaI and cloned into the polylinker of pT7.5 to give the construct pNR19, which was used as the template for the introduction of mutations into the signal peptidecoding portion of yacK. Site-specific mutations in pNR14 and pNR19 were constructed either by polymerase chain reaction methods or using the QuikChange TM system (Stratagene). All mutations were verified by DNA sequencing using the Applied Biosystems Prism TM BigDye TM Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems).
Pulse-Only and Pulse-Chase Experiments-An overnight culture of K38 containing both pGP1-2 (36) and the appropriate pT7 recombinant plasmid was grown aerobically in Luria-Bertani medium at 30°C with the appropriate concentrations of antibiotics (12). The strain was subcultured at a 1:80 dilution in fresh Luria-Bertani medium and grown until the A 600 nm reached 0.2. At this point 1 ml of culture was harvested and washed once in M9 medium (35). The washed cells were resuspended in 5 ml of M9 medium supplemented with 0.002% thiamine, 2 mM MgSO 4 , 0.1 mM CaCl 2 , and a methionine-and cysteine-free amino acid mix (0.01%). The cells were grown for a further hour at 30°C. During this period, and for the remainder of the experiment, the cells were incubated in 5 ml of M9 plus supplements in a 25-ml universal tube. Synthesis of the plasmid-encoded Tat-dependent precursor proteins, which are under the transcriptional control of the T7 10 promoter, was induced by a temperature shift from 30°C to 42°C for 15 min. This was followed by the addition of 400 g/ml rifampicin to inhibit the E. coli RNA polymerase. The samples were maintained at 42°C for a further 10 min and then transferred to 30°C for 20 min prior to labeling. 0.05 mCi of [ 35 S]methionine (NEN Life Science Products) was added to each 5-ml culture. For the pulse-only experiments the initial 0.5-ml sample was taken immediately. For the pulse-chase experiments the initial 0.5-ml sample was collected after a 5-min labeling period followed by the immediate addition of 750 g/ml unlabeled methionine. In both types of experiment additional 0.5-ml samples were removed at defined time points during the subsequent incubation. Samples were flash frozen in liquid nitrogen as they were collected. The samples were subsequently thawed and the cells pelleted by centrifugation. The pellets were resuspended in 75 l of SDS loading buffer, and the samples were analyzed by autoradiography following SDS-PAGE (37) on 12.5% acrylamide gels. Quantification of the gel bands was undertaken using a Fuji BAS-1000 PhosphorImager and the software package MacBAS version 2.0.
Subcellular Localization of Radiolabeled Proteins-The cells to be fractionated were prepared as for the pulse-chase experiments with the exception that only 2 ml of cell culture was labeled. After the appropriate chase period the cells were pelleted by centrifugation, resuspended in 1 ml of cell fractionation buffer (30 mM Tris-HCl, pH 8.0, 20% (w/v) sucrose, 1 mM Na 2 EDTA), and incubated at 20°C for 10 min. The cells were centrifuged, the supernatant discarded, and the pellet resuspended in 133 l of ice-cold 5 mM MgSO 4 . After 10 min on ice the cells were centrifuged, and the supernatant was retained as the periplasmic fraction.

RESULTS AND DISCUSSION
Establishing a Semiquantitative Export Assay-To assess the effect of mutagenesis of the twin arginine consensus region upon protein export it was first necessary to establish an appropriate assay. Since it has not so far proved possible to develop an in vitro transport assay for the bacterial Tat system we investigated methods for studying Tat translocation in whole cells. We chose the precursor of the E. coli protein SufI as the experimental substrate for our in vivo assay. SufI was originally identified on the basis that, when overproduced, it suppressed the cell division defect of an ftsI mutant (38). SufI is clearly homologous to the multicopper oxidase superfamily (39). Nevertheless, SufI is unlikely to bind copper cofactors because it contains only 2 of the 12 consensus copper-binding ligands, and neither of these is conserved in the SufI protein from the closely related organism Haemophilus influenzae. In addition, purified recombinant SufI does not contain detectable copper binding sites. 2 It is thus reasonable to expect that the observed export kinetics of SufI will not be complicated by the kinetics of cofactor insertion processes. The use of SufI as the assay substrate has a number of additional practical advantages. SufI is a sufficiently small protein that the mass difference between precursor (51.9 kDa) and mature (49.1 kDa) forms is readily detectable by denaturing electrophoresis, and therefore export can be routinely and conveniently monitored using a SDS-PAGE-based assay. SufI, in contrast to the majority of E. coli Tat substrates, is a water-soluble rather than peripheral membrane protein, which allows export to be confirmed by subcellular fractionation. Use of an indigenous substrate of the E. coli Tat pathway helps ensure that the experimental data obtained in our studies are relevant to the export of physiological substrates.
The twin arginine region of the SufI signal peptide (Fig. 1, top) exhibits a close match to the Tat consensus sequence differing only in conservatively substituting an isoleucine for the consensus leucine (this is the most frequently observed replacement at this position in Tat signal peptides; see Ref. 7) and substituting a glutamine for the consensus lysine residue. Two additional sequence features typical of bacterial signal peptides (6), a proline at the end of the h-region (Pro-22) and a basic residue in the c-region (Lys-24), are also present in the SufI signal sequence.
A plasmid, pNR14, was constructed in which expression of sufI is under the control of the T7 10 promoter. Upon provision of T7 RNA polymerase, and in the presence of rifampicin to block transcription from E. coli promoters, this construct allows specific in vivo radiolabeling of the SufI precursor protein.
A pulse-chase experiment using the sufI-bearing plasmid pNR14 is shown in Fig. 1a. After induction of T7 polymerase coded by the helper plasmid pGP1-2, the cells were pulse labeled with [ 35 S]methionine, followed by the addition of excess unlabeled methionine. Over time a labeled protein with the apparent molecular mass of pre-SufI is converted to a smaller mature form. Neither form is present when a plasmid lacking the sufI insert is used (not shown), confirming that these proteins correspond to precursor and processed forms of SufI. Subcellular fractionation experiments showed that the mature form represents protein that has been exported to the periplasm (Fig. 1b, lanes 2 and 3).
A strain bearing a deletion in the tatC gene has been shown previously to be completely defective in the correct localization of proteins with Tat signal peptides (13). When the pulse-chase experiment was repeated in this ⌬tatC background only the precursor protein was visible after a 60-min chase (Fig. 1b, lane  1), indicating that processing of pre-SufI is exclusively Tat-dependent and that no export of this protein occurs via the Sec pathway.
Although export of wild-type precursor is essentially complete within minutes (Fig. 1a), precursors with mutated signal peptides were anticipated to have slowed rates of export. It was therefore important to establish that export continued to occur for extended time periods under our assay conditions. In the experiments shown in Fig. 1c, labeling of newly synthesized pre-SufI was initiated by the addition of [ 35 S]methionine and allowed to continue throughout the experiment. The mature protein continued to accumulate over the full 60-min time period, demonstrating that export occurs throughout the time scale of the experiment.
Analysis of Site-specific Amino Acid Substitutions in the SufI Signal Peptide-A range of site-specific amino acid substitutions was introduced into the signal peptide coding region of sufI. The resulting engineered precursors have been given shorthand designations made up of the substituted amino acid type and position followed by the replacement amino acid. Thus precursor SufI-R5K carries a lysine substitution of the arginine at position five in the SufI signal peptide. The effects of the various site-specific mutations on the export kinetics of pre-SufI were assessed by pulse-chase assays. The averaged export data for a minimum of three independent cultures expressing each engineered precursor are plotted in Fig. 2. Fractionation studies were used to verify that the mature protein observed in the pulse-chase experiments was located in the periplasm (data not shown). These experiments confirm that the slow processing exhibited by some of the signal peptide mutants represents a genuine export event and is not a consequence of nonspecific proteolysis of the unexported precursor protein. No processing was observed over a 60-min chase for any of the mutant precursors when the export experiments were repeated in a ⌬tatC background (data not shown). Thus, none of the signal peptide mutations investigated in this study caused the precursor protein to default onto the Sec export pathway. This phenotype may not, however, reflect the true potential for interactions between the altered signal peptides and the Sec apparatus but may instead originate from an incompatibility between the Sec pathway and the mature SufI protein. Indeed, there is now clear evidence that some Tat pathway proteins are inherently refractory to transport by the Sec apparatus (20,24).
Mutations Affecting the Twin Arginine Residues-The invariant arginine residues of pre-SufI were conservatively replaced both individually and in combination by the amino acid lysine. The individual arginine to lysine mutations severely retard export of pre-SufI, whereas export was blocked completely when both arginines were replaced (Fig. 2a). These results are in marked contrast to the behavior of Sec signal peptides where mutations that conserve the n-region charge have no effect on the transport process (40) and argue that both Tat consensus arginine residues play a specific and critical role in mediating the interaction between the signal peptide and the Tat apparatus. That the single lysine for arginine mutations in the SufI signal peptide still permits transport by the Tat pathway was surprising because equivalent mutations had been found to block both the Tat-dependent export of Zymomonas mobilis glucose-fructose oxidoreductase (29) and the import of wheat pre-23K protein via the plant thylakoid Tat system (16). However, a further study using a Tat signal peptide:␤-lactamase chimera expressed in E. coli suggested that the first consensus arginine residue could be replaced not only by lysine but also by glutamate, valine, or isoleucine without completely blocking export (26). It should be kept in mind though that there is no evidence that the transport observed in this early study was mediated by the Tat pathway. These variations in the severity of a single lysine for Tat consensus arginine substitutions observed in different studies could be thesis and processing were assessed by SDS-PAGE of whole cells followed by autoradiography. The mean percentage of total SufI protein remaining in precursor form in whole cells at each of the indicated time points is plotted (n ϭ 3-6). The bars represent the standard error of the mean. Panel a, the export of wild-type pre-SufI (q) is compared with that of precursors with alterations in the Tat signal peptide invariant arginine pair: SufI-R6K (E), SufI-R5K (f), SufI-R5A (OE), and SufI-R5K,R6K (Ⅺ). Panel b, the export of wild-type pre-SufI (q) is compared with that of precursors with substitutions of the Tat signal peptide consensus serine residue, SufI-S4A (E) and SufI-S4C (f), as well as the double mutant SufI-S2A,S4A (Ⅺ). Panel c, the export of wild-type pre-SufI (q) is compared with that of precursors with various Tat signal peptide mutations: SufI-Q7L (OE), SufI-I9A (Ⅺ), SufI-K24A (E), and SufI-K24D (f). Panel d, the export of wild-type pre-SufI (q) is compared with that of precursors with substitutions of the Tat signal peptide consensus phenylalanine residue: SufI-F8L (E), SufI-F8Y (f), and SufI-F8A (Ⅺ). Panel e, the export of wild-type pre-SufI (q) is compared with that of precursors with substitutions of the glutamine that occupies the position where the Tat signal peptide consensus has a lysine residue: SufI-Q10L (E), SufI-Q10A (f), and SufI-Q10K (Ⅺ).
indicative of genuine differences in the behavior of disparate signal peptides. Alternatively, they could be a consequence of the variable sensitivity of the export assays employed by different investigators. In an attempt to distinguish between these two possibilities we produced an additional lysine for arginine mutation in another E. coli Tat substrate, pre-YacK, a multicopper oxidase (39) of unknown physiological function. YacK is homologous to SufI (37% amino acid sequence identity), and the two proteins have quite similar Tat signal peptides (Figs. 1 and 3). A pulse-chase export assay analogous to that used for pre-SufI was established for pre-YacK with the processed mature form of YacK shown to be located in the periplasm (Fig. 3a, lanes 2 and 3), and export demonstrated to be tatC-dependent (Fig. 3a, lane 1) and to continue for at least 60 min after labeling was initiated (data not shown). Since export of the YacK and SufI was analyzed by identical methodology, the phenotypes of equivalent mutations in the two proteins are directly comparable. Whereas single arginine to lysine mutations in pre-SufI permit export, it was found that an analogous mutation in YacK (YacK-R3K, Fig. 3b) abolishes transport. We conclude that in certain signal peptide contexts the presence of both arginine residues is not an obligate requirement for transport by the Tat pathway. Nevertheless, even in such a permissive signal peptide it is likely that an arginine can only be functionally replaced with another positively charged amino acid (that is lysine) because we found export to be blocked by an alanine for arginine substitution (SufI-R5A, Fig. 2a). This observation suggests that the Tat system recognizes the twin arginine residues in part via electrostatic interactions.
Mutations Affecting the Consensus Serine Residue-We have noted previously (7) that the residue that directly precedes the arginine pair is most frequently one of those amino acids (serine, threonine, asparagine, and aspartate) that is a preferred capping residue for the NH 2 termini of helices (41). As a consequence we speculated that this residue might stabilize the core of the signal peptide in an ␣-helical conformation. We tested the possible importance of the SufI Tat consensus serine as a helix cap by replacing this residue with alanine, an amino acid with a high helical propensity, but which is unable to stabilize the NH 2 termini of helices because it does not have a hydrogen bond-accepting side chain. This SufI-S4A mutation had no significant effect on export kinetics (Fig. 2b), suggesting that neither the functionality of the serine side chain nor the presence of a helix-capping residue at this position is critical for the export process. NH 2 -terminal to the consensus serine the SufI signal peptide possesses an additional good helix-capping residue, Ser-2. This residue could potentially substitute for the function of the consensus serine in the SufI-S4A mutant. We were able to exclude this possibility by demonstrating that export of a mutant lacking both serine residues was not significantly impaired (SufI-S2A,S4A; Fig. 2b). The consensus serine residue in the SufI signal peptide was additionally mutated to cysteine, an amino acid that differs structurally from serine only in replacing the ␥ oxygen atom with a sulfur atom. The resultant SufI-S4C construct exhibited a substantially slowed rate of export (Fig. 2b). Given the unchanged transport phenotype of the SufI-S4A mutant where all side chain functionality is missing, it is most likely that it is the substantially increased hydrophobicity of the cysteine side chain in SufI-S4C which is interfering with the export process.
Mutations at the Consensus "X," Phenylalanine or Leucine Positions-A feature of the consensus motif, noted here for the first time, is that the amino acid position immediately following the paired arginine residues (residue X in the consensus motif) is any polar residue or, rarely, the short side chain amino acids glycine or alanine. To test the importance of this amino acid to Tat transport we changed the glutamine found at this position in the SufI signal peptide to leucine, which has a similarly sized, but highly hydrophobic, side chain. This SufI-Q7L mutation had no significant effect on export rate (Fig. 2c), and we therefore conclude that the polar amino acid that follows the twin arginine residues is not critical for Tat transport.
The consensus phenylalanine residue is, after the invariant arginine residues, the most highly conserved amino acid of the bacterial Tat motif, being found in approximately 80% of twin arginine signal peptides. Remarkably, a phenylalanine is never found at this position in plant Tat transfer peptides. We found that replacing the consensus phenylalanine of pre-SufI with either tyrosine (SufI-F8Y), which differs from phenylalanine by having a hydroxyl group attached to the carbon of the aromatic ring, or with alanine (SufI-F8A), which effectively removes the side chain functionality, severely retards export (Fig. 2d). In contrast, a substitution by leucine, the amino acid found most frequently after phenylalanine at this position, had no discernible effect on export kinetics (SufI-F8L, Fig. 2d). The nature of the amino acid side chain at the phenylanine consensus position is thus critical to export efficiency. The fact that leucine but not tyrosine can be substituted for the consensus phenylalanine argues against the aromatic nature of the phenylalanine side chain being a major arbiter of interactions with the Tat apparatus. Instead, we suggest that it is the high hydrophobicity (and possibly also high helical propensity) exhibited by phenylalanine and leucine, but not tyrosine, which is the critical structural determinant at this consensus position.
The amino acid position three residues after the arginine pair is always hydrophobic with a strong preference for a leucine residue. Plant Tat transfer peptides also contain a hydrophobic amino acid at the equivalent position, and this has been shown experimentally to have a weak influence on transport (42). The importance of a nonpolar side chain at the consensus leucine position in bacterial Tat signal peptides was tested by substituting an alanine residue for the isoleucine present in the signal peptide of pre-SufI. This SufI-I9A mutation showed a small decrease in the rate of export (Fig. 2c), suggesting that, as for thylakoid Tat signal peptides, a hydrophobic amino acid at the consensus leucine position contributes to efficient Tat transport.
Mutations at the Consensus Lysine Position-A lysine residue is normally present four amino acids after the arginine pair in bacterial Tat signal peptides. This is in marked contrast to thylakoid Tat transfer peptides where a charged amino acid is never found at this position. Nevertheless, bacterial Tat signal peptides containing the consensus lysine are capable of mediating thylakoid import via the chloroplast Tat pathway (17,18). In the SufI signal peptide the consensus lysine is replaced by a glutamine residue. To investigate the significance of this substitution we mutated the glutamine to the consensus lysine. Surprisingly, export of the resultant SufI-Q10K mutant was severely retarded (Fig. 2e). We also investigated the effects of replacing the glutamine with alanine (SufI-Q10A) to remove the side chain functionality at this position, or leucine (SufI-Q10L), an amino acid with a hydrophobic side chain of similar bulk to that of glutamine. Both of these substitutions resulted in only small decreases in the rate of SufI export rather than the severe transport defect exhibited by the SufI-Q10K mutant (Fig. 2e). Thus for SufI the identity of the amino acid at the consensus lysine position appears to be functionally important, but, surprisingly, of the amino acids tested it was substitution with the consensus lysine residue which had the strongest inhibitory effect on export. We decided to investigate this unusual finding further by examining the effect of mutating the consensus lysine in YacK. Replacing the lysine with either alanine or arginine did not slow export (YacK-K8A and YacK-K8R, Fig. 3b), confirming that the lysine is not required for efficient export. In fact the conservative YacK-K8R mutant showed a small increase in export rate, providing some support for the idea that the consensus lysine may function to retard export. At the current time the rationale for such behavior is speculative. Perhaps, given that thylakoid proteins and SufI lack both cofactors and the consensus lysine, the lysine might mediate interactions with proteins which ensure that the cofactor has been inserted before export is attempted (8).
Investigating the Role of the c-Region Charge in Tat Pathway Export-The basic residues characteristically found in the cregion of thylakoid Tat signal peptides are not required for Tat targeting but act to prevent functional interaction of the precursor protein with the Sec apparatus (24). Bacterial Tat signal peptides, including that of pre-SufI, also commonly possess a positively charged c-region (18,43). We found that mutation of the lysine found in this region of pre-SufI either to alanine or to aspartate had no discernible effect on the rate of export (SufI-K24A and SufI-K24D, Fig. 2c), while transport remained exclusively Tat-dependent. As noted above, the SufI mature region may well be Sec-incompatible, and this would mask our ability to assess the Sec compatibility of the signal peptide itself. Clearly, however, our results show that a c-region basic residue is not essential for transport of an authentic bacterial Tat substrate through the Tat apparatus.
Although the c-region of Tat signal peptides commonly contains basic amino acids, we note here that acidic residues are rarely found in the Tat signal peptides of either bacteria or plants and are in fact totally absent from E. coli Tat substrates. The net effect is that the c-regions of bacterial Tat signal peptides have an overall positive charge (the mean charge is ϩ0.64 when calculated from the non-Rieske protein data set in Ref. 7). In our SufI-K24D mutant the signal peptide c-region has both an acidic residue and an overall negative charge (note also that the first charged residue in the mature protein is at residue 16). Nevertheless, this protein exhibits normal export by the Tat pathway (Fig. 2c), indicating that the c-region charge is not critically important for export.
Concluding Comments-In summary, our studies establish the importance of the invariant arginine residues of Tat signal peptides for the efficient export of authentic E. coli Tat substrates. However, we find that in certain signal peptide contexts transport is still possible when lysine is substituted for one of the consensus arginine residues, and therefore paired arginine residues are not an absolute requirement for Tat signal peptide function. What then is the selective pressure that causes retention of the arginine pair? Pre-SufI mutants with a single lysine for arginine substitution, although competent for transport by the Tat pathway, have transit half-times that greatly exceed the maximum rate of cell division in E. coli. It must, therefore, be questionable whether Tat signal peptides with such substitutions would be physiologically viable, and this may be the rationale for the evolutionary retention of an arginine pair. Our experiments also establish that efficient export of bacterial Tat substrates depends strongly on the consensus phenylalanine residue. This observation, together with the deleterious effects of certain substitutions at other consensus positions, strongly supports the idea that the observed sequence conservation of the extended twin arginine motif found in bacterial, but not plant, signal peptides reflects an important role for these residues in mediating specific interactions with Tat pathway components.