a fundamental event 
 in the folding and oligomerization of membrane 
 proteins is the association of the transmembrane ( tm ) helices .
helix 
 association is required to achieve the final fold and oligomeric state 
 of the protein . a favorite system for investigating the rules that 
 govern tm helix association are the single - span membrane proteins , primarily because a variety of methods are available for measuring 
 their oligomerization ( including fret , sedimentation equilibrium analytical 
 ultracentrifugation , in vivo assays in biological 
 membranes , sds - page , and steric trapping ) .
conversely , assessing the folding energetics of multispan membrane 
 proteins still represents a tremendous challenge .
in addition to being a tractable system , the single - span membrane 
 proteins attract interest because of their biological importance . 

these proteins comprise the most numerous class of membrane proteins , 
 constituting about half of the total . rather than acting as 
 mere membrane anchors for soluble domains ,
as it was once assumed , 
 the oligomerization of single tm domains actively plays roles in assembly , 
 signal transduction , ion conduction and regulation in a wide variety 
 of biological processes . to investigate 
 the basis of oligomerization in tm helices , our 
 group and others have pursued a strategy based on the analysis of 
 frequently occurring association motifs .
one of the most important motifs is gasright ( figure 1 ) , which is 
 best known as the fold of a widely studied model system for tm association , 
 the glycophorin a tm dimer .
gasright gets its name from its right - handed crossing angle ( figure 1b ) , and from the characteristic small amino acids 
 at its interface ( gas : gly , ala , ser ) , which are arranged to form gxxxg and gxxxg - like patterns ( gxxxa , 
 axxxg , etc . ) . in many ways
gasright parallels the important coiled coil , a frequently occurring interaction 
 motif and model for folding and association for soluble proteins . like the coiled coil
, gasright is characterized by a 
 specific geometry ( a short interhelical distance and a crossing angle 
 near 40 ) , it has a distinctive sequence signature ( the 
 gxxxg patterns ) , and is one of the most common oligomerization motifs , 
 if not the most common . structural features of 
 the gasright tm association motif . 

( a ) the gasright motif ( which is best known as the fold 
 of the tm region of glycophorin a ) is a right - handed helical dimer 
 with a short interhelical distance d and a right - handed 
 crossing angle  of approximatively 40. the gxxxg 
 sequence pattern near the crossing point ( marked in red in the green 
 helix ) allows the backbones to come into close contact .
( b ) the contact 
 enables the formation of networks of interhelical hydrogen bonds between 
 ch donors and carbonyl oxygen acceptors ( shown in detail 
 in ( c ) ) . in a recent computational analysis 
 of transmembrane dimer geometry
, 
 we proposed that the primary role of gxxxg in gasright is 
 to promote the formation of networks of stabilizing hydrogen bonds 
 between ch donors and carbonyl oxygen acceptors on 
 opposed helices ( figure 1b , c ) .
more specifically , we proposed that the small amino 
 acids perform two distinct functions : the first is to create permissive 
 steric conditions , allowing the two helices to come in backbone contact , 
 thus bringing the ch donors and carbonyl acceptors 
 in proximity
. the second function , which is performed exclusively 
 by gly , is to increase the number of hydrogen bonds by donating with 
 the second h , which corresponds to the side chain r - group in 
 all other amino acids . to perform these functions ,
the formation 
 of this network of hydrogen bonds is also dependent on the specific 
 crossing angle of gasright ( 40 ) , which precisely 
 aligns ch donors spaced at i , i+1 on one helix against carbonyl acceptors spaced at i , i+3 on the opposing helix ( see figure 
 4 in mueller et al . ) .
carbons are generally weak donors , but the c 
 in proteins is activated by the electron - withdrawing amide groups 
 on both sides , and quantum calculations indicate that the energy of 
 ch hydrogen bonds may be as much as one - third to half 
 of that of canonical donors in vacuum . therefore , 
 they are likely to be stabilizing factors in proteins embedded in 
 the hydrophobic milieu of the membrane , particularly when they occur 
 in multiple instances at the same interface , as in the gasright motif ( figure 1 ) . an ir - based investigation of the cd2 stretching mode 
 of a ch donor in the transmembrane domain of glycophorin 
 a produced an estimated contribution of 0.88 kcal / mol for 
 the hydrogen bond .
conversely , a folding 
 study of the multispan membrane protein bacteriorhodopsin in which 
 a cho side chain hydroxyl acceptor 
 ( thr-24 ) was mutated indicated that this particular bond was not stabilizing .
subsequent computational work suggested that 
 the orientation of the groups can determine whether an interaction 
 may be strongly favorable or unfavorable .
the exact contribution of hydrogen bonds to membrane protein 
 folding 
 and association  whether the donor is a ch 
 or a more  canonical  n  h or o  h group 
  is still unresolved .
a governing 
 assumption maintains that donors and acceptors buried in the membrane 
 would not pay a significant desolvation penalty upon helix association , 
 and therefore the formation of hydrogen bonds should contribute appreciably 
 to the stability of membrane proteins .
yet , the limited number of experimental 
 observations made to date seem to indicate that the contribution of 
 hydrogen bonding in the membrane may be , surprisingly , of the same 
 magnitude observed for water - soluble proteins . despite the scarce experimental evidence regarding 
 the contribution 
 of ch hydrogen bonds to tm interactions , the hypothesis 
 that they drive folding and oligomerization remains compelling . in 
 particular
, the fact that the prevalent gasright motif 
 corresponds to the only interhelical geometry that maximizes formation 
 of cho = c networks , strongly 
 suggests that these bonds are indeed a major contributor to association . under these premises
, we hypothesized that a 
 computational structural search based on the simultaneous optimization 
 of side chain packing and ch hydrogen bonding may be 
 able to predict the structure of gasright dimers .
the resulting 
 program , named catm , was tested against the small database of known 
 gasright homodimeric structures .
the finding provides further indirect support that ch 
 hydrogen bonding is likely to be a structural determinant of gasright dimers .
the positive result 
 also indicates that catm may be a powerful tool for assisting the 
 experimental investigation of gasright homodimers of unknown 
 structure . to test the ability of our methods to predict ab 
 initio the structure 
 of unknown gasright dimers ,
here we investigate adck3 , 
 a human mitochondrial protein that is a member of the highly conserved 
 ubib protein kinase - like family .
ubib 
 family members account for approximately one - quarter of microbial 
 pkl sequences , are ubiquitous among eukaryotes , and are strongly associated with lipid metabolism .
most organisms have a ubib family member 
 that is required for the biosynthesis of coenzyme q ( coq , ubiquinone ) .
deletion of the escherichia 
 coli gene ubib(54 ) or the yeast gene coq8(55 ) completely halts coq biosynthesis .
similarly , 
 mutations to human adck3 are known to cause coq deficiency and cerebellar 
 ataxia , and mutations to human adck4 were recently shown to cause coq deficiency 
 and a steroid - resistant nephrotic syndrome .
our knowledge of the molecular mechanism by which ubib family proteins 
 enable coq biosynthesis is limited , primarily because the endogenous 
 substrates of ubib proteins have not yet been discovered . however , 
 we do know that coq8p in yeast somehow stabilizes a complex of coq 
 biosynthesis enzymes .
coq biosynthesis 
 occurs within the context of cellular membranes , either the plasma 
 membrane of prokaryotes or the inner mitochondrial membrane of eukaryotes , 
 and the responsible enzymes are either integral membrane proteins 
 or peripherally associated membrane proteins .
adck3 , which contains a predicted tm domain , is also likely to associate 
 with membranes , but this hypothesis has not yet been tested . biochemical 
 characterization of the adck3 tm domain would provide an important 
 foundation for understanding how it enables coq biosynthesis .
the potential functional importance of the adck3 tm region is underlined 
 by the existence of a mutation at the putative edge of the tm domain 
 ( r213w ) that disrupts coq biosynthesis and causes cerebellar ataxia 
 in human patients .
furthermore , dimerization 
 of single - span tm domains is known to be central to the regulation 
 of some kinase families , such as the receptor tyrosine kinases .
however , it was unknown whether the predicted tm helix of adck3 
 could actually insert into biological membranes and whether the tm 
 helix can self - associate to potentially drive dimerization of adck3 . here , we demonstrate experimentally that the tm domain of adck3 
 inserts into membranes and self - associates . using extensive mutagenesis
, 
 we also show that the interaction interface is consistent with the 
 structural models predicted by catm , which involves an extended gly - zipper 
 motif ( i.e. , a series of gly amino acids 
 separated at i , i+4 ) .
the experimental 
 and computational data also indicates that the gly - zipper interface 
 is potentially compatible with alternative conformations of the tm 
 domain , opening the possibility that conformational changes of the 
 tm dimer may be important for adck3 function .
all oligonucleotides were purchased 
 in desalted form from integrated dna technologies and used without 
 purification .
the expression vectors pcckan , pccgpa - wt , and pccgpa - g83i , 
 and male deficient e. coli strain 
 mm39 were kindly provided by dr .
the genes encoding the tm domain of adck3 ( 214-lanfgglavglgfgala-230 ) 
 and adck4 ( 92-lanfgglavglglgvla-108 ) were cloned into 
 the nhei - bamhi restriction sites of the pcckan vector . 

site directed mutations to produce single amino acid variants in the 
 tm domain of adck3 were introduced with the quikchange kit ( stratagene ) .
a freshly streaked colony 
 was inoculated into 3 ml of lb broth containing 100 g / ml ampicillin 
 and grown overnight at 37 c .
30 l of overnight cultures 
 were inoculated into 3 ml of lb broth and grown to an od420 of approximately 0.81.1 ( od600 of 0.4 to 0.6 ) 
 at 37 c . after recording the optical density ,
1 ml of cells 
 was spun down for 10 min at 17000 g and resuspended 
 in 500 ml of sonication buffer ( 25 mm tris - hcl , 2 mm edta , ph 8.0 ) . 

cells were lysed by probe sonication at medium power for 8 s over 
 ice .
an aliquot was removed from each sample and stored in sds - page 
 loading buffer for immunoblotting .
the lysates were then cleared by 
 centrifugation at 17000 g , and the supernatant was 
 kept on ice for chloramphenicol acetyltransferase ( cat ) activity assay . to confirm proper membrane 
 insertion and orientation of the toxcat constructs , overnight cultures 
 were plated on m9 minimal medium plates containing 0.4% maltose as 
 the only carbon source and grown at 37 c for 48 h. cat activity
briefly , 1 ml of buffer containing 0.1 mm acetyl coa , 0.4 mg / ml 
 5,5-dithiobis(2-nitrobenzoic acid ) or ellman s reagent , 
 and 0.1 m tris - hcl ph 7.8 , were mixed with 40 l of cleared 
 cell lysates and the absorbance at 412 nm was measured for 2 min to 
 establish basal enzyme activity rate .
after addition of 40 l 
 of 2.5 mm chloramphenicol in 10% ethanol , the absorbance was measured 
 for an additional 2 min to determine cat activity .
the basal cat activity 
 was subtracted and the value was normalized by the cell density measured 
 as od420 .
all measurements were determined at least in 
 duplicate and the experiments were repeated at least twice .
the cell lysates ( 10 l ) 
 were loaded onto a nupage 412% bis - tris sds - page gel ( invitrogen ) 
 and then transferred to pvdf membranes ( vwr ) for 1 h at 100 millivolts . 

blots were blocked using 5% bovine serum albumin ( us biologicals ) 
 in tbs - tween buffer ( 50 mm tris , 150 mm nacl , 0.05% tween 20 ) for 
 2 h at 4 c , incubated with biotinylated anti - maltose binding 
 protein antibodies ( vector laboratories ) overnight at 4 c , followed 
 by peroxidase - conjugated streptavidin ( jackson immunoresearch ) for 
 2 h at 4 c .
blots were developed with the pierce ecl western 
 blotting substrate kit and chemiluminescence was measured using an 
 imagequant las 4000 ( ge healthsciences ) .
the structure of adck3-tm was 
 predicted with catm , which is distributed 
 with the open source msl c++ library v. 1.2 at http://msl-libraries.org .
the computational mutagenesis 
 was performed on all adck3 models by applying the same point mutations 
 measured experimentally in the context of a fixed backbone , followed 
 by side chain optimization .
side chain mobility was modeled using 
 the energy - based conformer library applied at the 95% level .
energies were determined using the charmm 22 
 van der waals function and the hydrogen 
 bonding function of scwrl 4 , as implemented 
 in msl , with the following parameters 
 for c donors , as reported previously : b = 
 60.278 ; d0 = 2.3  ; d = 1.202  ; max = 74.0 ; max = 98.0. the relative 
 energy of each mutant was calculated aswhere ewt , dimer and emut , dimer are the energies of the 
 wild type and mutant sequence , respectively , in the dimeric state , 
 and ewt , monomer and emut , monomer are the energies of the wild type and mutant 
 sequence , respectively , in a side chain optimized monomeric state 
 with the same sequence . as reported previously ,
the effect of each mutation was classified in four categories 
 ( analogous to the experimental mutagenesis ) using the following criterion : 
 category 0 ,  wt - like  , emut < 2 kcal / mol ; category 1 ,  mild  , 2  
 emut < 4 ; category 2 ,  severe  , 
 4  emut < 8 ; category 
 3 ,  disruptive  , emut  8 .
the numerical category values were averaged to calculate 
 the average position - dependent disruption value .
the protein kinase - like 
 domain of adck3 is preceded on the n - terminal side by a region of 
 undefined function .
a predicted tm helix within this region is annotated 
 in uniprot for the close homologue adck4 , providing a potential anchor for the protein at the inner mitochondrial 
 membrane ( figure 2a ) .
uniprot does not report 
 a predicted tm domain for the corresponding region of adck3 , but the 
 sequence of the putative tm segment is highly conserved between the 
 two proteins .
the same general domain organization and function is 
 also predicted for the yeast homologue coq8p . given that adck3 and 
 adck4 are localized to the mitochondrial matrix , the tm domain would position their catalytic kinase domains 
 on the matrix face of the inner membrane , the same localization of 
 the enzymes involved in the biosynthesis of coenzyme q. therefore , it is important to verify the tm domain experimentally 
 and to investigate its potential functional role .
( a ) domain organization of adck3 homologues , which are proteins associated 
 with the mitochondrial inner membrane .
they are predicted to contain 
 a tm domain ( yellow ) and a protein kinase - like domain ( white ) .
( b ) 
 the sequence alignment of the tm domains of adck3 , adck4 ( yellow box ) . 

the tm domains of adck3 and adkc4 , which differ only at two positions , 
 contain a number of gxxxg - like motifs , including an extended gly - zipper 
 motif ( red ) and a second axxxg motif which is off - register by two 
 positions ( magenta ) .
( c ) sequence logo of the alignment of 400 sequences 
 homologous to adck3 from a broad range of eukaryotic species highlights 
 conservation in the tm domain and in the n - terminal side of the juxta - membrane 
 region .
all gly positions in the gly - zipper ( red ) appear strongly 
 conserved .
identifiers of the sequences used for the alignment are 
 provided in supplementary text s1 ( si ) .
the sequences of the putative 
 tm domains of adck3 and adck4 are 
 aligned in figure 2b .
as summarized in table 1 , these sequences have low hydrophobicity and a 
 relative short length ( 17 amino acids ) , and thus are not well recognized 
 by prediction servers .
the tm domain of adck3 , which contains one 
 polar amino acid ( asn 216 ) , is recognized as a borderline tm sequence 
 by most servers .
specifically , the segment is not recognized by tmhmm and e(z ) , but the 
 segment is predicted as transmembrane by membrain and hmmtop , and phobius and g prediction recognize it with low confidence .
adck4 shares over 50% sequence 
 identify with adck3 but their tm domains are almost identical , differing 
 only at two positions ( figure 2b ) . because 
 of these two substitutions ( and primarily because of the a228 v substitution ) , 
 the hydrophobicity of the tm domain of adck4 is higher ( as calculated 
 with either the wimley - white octanol scale or the  biological  scale ) and is sufficient to be predicted by most servers , except e(z ) , 
 with good confidence ( table 1 ) .
wimley  white 
 octanol scale 
 ( kcal / mol ) . biological hydrophobicity scale 
 ( kcal / mol ) .
g predictior at http://dgpred.cbr.su.se ( in paretheses 
 the gapp for the predicted tm segment , 
 kcal / mol ) .
it is important to consider 
 that tm prediction servers are trained against a majority of proteins 
 that are inserted in the membrane via a translocon mediated mechanism . 

the sequence requirements for translocon mediated insertion in an 
 eukaryotic system are well understood .
a recent analysis 
 in a bacterial system shows good overall correspondence to the mammalian 
 system , but the hydrophobicity threshold appears to be distinctly 
 lower .
much less is known about the requirements 
 for membrane insertion of mitochondrial integral membrane proteins 
 that are encoded in the nucleus , such as adck3 .
there is , however , 
 good indication that the hydrophobicity threshold for these proteins 
 should be even lower , to avoid mistargeting of these proteins to the 
 endoplasmic reticulum and to facilitate their translocation to the 
 mitochondrion .
based on the above considerations , it is highly probable that the 
 predicted tm segments of adck3 and adck4 are indeed bona fide tm domains .
as 
 shown in figure 2b , the predicted tm regions 
 of adck3 and adck4 are very rich in small amino acids such as gly , 
 ala and ser ( 9 in each ) .
the sequences contain a number of gxxxg and 
 gxxxg - like ( axxxg ) helix association patterns , which appear to be 
 evolutionarily conserved ( figure 2c ) .
in particular , 
 they contain an extended gly - zipper motif , i.e. , a series of small amino acids ( 215-axxxgxxxgxxxg-227 ) spaced 
 at i , i+4 , highlighted in red in 
 figure 2b .
they also contain an additional 
 axxxg motif ( magenta ) , which is off - frame by two positions with respect 
 to the gly - zipper .
they occur with high frequency 
 in tm helices , both in multispan proteins 
 and in oligomerizing single - span membrane proteins , and are often important for biological function .
the presence of gxxxg - like motifs in the putative 
 tm sequence of adck3 raised the question of whether this domain oligomerizes . 
 to investigate this question
, we analyzed the sequence with catm , a program for the structural prediction of gasright motifs , an important and common class of gxxxg - mediated 
 dimers .
as shown in figure 3 , catm predicts five alternative models for the 
 tm sequence of adck3 .
the position of the crossing point between 
 the two helices is marked ( dot ) , and the interfacial positions that 
 surround this crossing point are highlighted by a green parallelogram . 

all the positions that are involved in intermonomer contacts at the 
 interface are highlighted in either yellow , or in red if they belong 
 to the gly - zipper motif .
the scores of the top models of adck3 in 
 catm ( 59.8 , 50.8 , and 47.7 for models 1 , 2 , 
 and 3 respectively ) are comparable to the scores obtained for the 
 five known structures of gasright motifs ( which range between 
 56 and 38 ) , which catm is able to predict at near 
 atomic precision .
catm predicts multiple 
 modes of interaction along the gly - zipper 
 motif of adck3 .
the four positions that surround the 
 crossing point are marked by a green parallelogram and are underlined 
 in the sequence .
the positions involved in interhelical packing at 
 the dimer interface are highlighted : in red are the interfacial positions 
 that belong to the extended gly - zipper motif of adck3 ; all other interfacial 
 positions are highlighted in yellow .
the table summarizes the geometry 
 of the five models : interhelical distance d ; crossing 
 angle  ; vertical ( z ) and axial (  ) 
 coordinates of the crossing point within the parallelogram of closest 
 approach ; and energy score e. for the geometric definitions , 
 see figure s2 ( si ) and mueller et al .
model 1 
 and 2 are related geometries whose crossing points fall in the quadrilateral 
 defined by gly 219 , leu 220 , gly 223 and leu 224 ( axxxglxxglxxg ) . 

these two models differ by the position of the crossing point and , 
 most importantly , by their crossing angle , which is near the canonical 
 40 of gasright motifs for model 2 , and narrower 
 for model 1 ( 27.1 ) .
the smaller crossing angle causes 
 model 1 to have a more extended interface , which is reflected also 
 by the more extensive van der waals interaction of model 1 .
both models 
 have 12 interhelical ch hydrogen bonds , although model 
 2 has a better overall hydrogen bonding score ( tables s1 and s2 ( si ) ) .
the other three predicted models 
 cross at different sections of 
 the gly - zipper .
model 3 and model 5 are variations that cross within 
 the n - terminal side of the zipper ( anxxglxxgxxxg ) .
conversely , 
 model 4 crosses on the c - terminal side of the zipper ( axxxgxxxglxxga ) .
catm does not produce any model mediated by the off - frame 
 axxxg motif of adck3 ( magenta in figure 2b ) . 

the coordinates of all adck3 models are available as supporting information and for download at http://seneslab.org/adck3_models . to investigate the structural predictions of 
 catm
, we assessed the dimerization of adck3-tm and adck4-tm experimentally 
 using toxcat , a widely used assay for tm association in biological 
 membranes .
this assay involves the biological 
 expression in the membrane of e. coli of a chimeric 
 construct that fuses the tm domain of interest with the toxr transcriptional 
 activator of vibrio cholera ( figure 4a ) .
tm helix association 
 leads to the dimerization of the toxr domain , resulting in expression 
 of the reporter gene chloramphenicol acetyltransferase ( cat ) . the 
 expression level of cat ( measured by its enzymatic activity )
is compared 
 to that of a stable dimer , glycophorin a ( gpa ) , and to a monomeric 
 gpa variant ( gpa - g83i ) as standards . adck3-tm and adck4-tm associate strongly 
 in toxcat .
( a ) toxcat 
 is an in vivo assay based on a construct in which the transmembrane 
 domain under investigation is fused to the toxr transcriptional activator 
 of v. cholerae .
transmembrane association results 
 in the expression of a reporter gene in e. coli cells , 
 which can be quantified .
the toxcat construct containing the tm domain of adck3 and 
 adck4 can use maltose as a carbon source , demonstrating correct insertion . 

gpa : glycophorin a positive control ; no tm : pcckan plasmid without 
 tm insert , negative control .
adck3 shows approximately 150% of the cat activity of the strong transmembrane 
 dimer of glycophorin a ( gpa ) .
the monomeric g83i mutant ( gpa * ) is 
 used as a negative control .
we first tested whether the constructs inserted correctly 
 in the 
 plasma membrane of e. coli , using a complementation 
 test in the male deficient strain mm39 .
the adck3-tm 
 and adck4-tm toxcat constructs supported growth in minimal media with 
 maltose as the sole carbon source ( figure 4b ) , indicating that the fusion proteins are recognized as tm domains 
 and are expressed in the bacterial inner membrane in the correct orientation , 
 with the mbp moiety positioned on the periplasmic side . to examine 
 whether adck3 oligomerizes in toxcat , we quantified 
 the enzymatic activity of the reporter gene cat , as an indirect measure 
 of its expression .
as shown in figure 4c , the 
 cat activities of the adck3-tm and adck4-tm constructs are higher 
 than the activity of the gpa standard , which is a stable homodimer . 

these results indicate that the tm domain of adck3 and adck4 form 
 strong homo - oligomers in toxcat . to assess experimentally the 
 interaction interface of the adck3-tm oligomer and validate the computational 
 predictions , we performed large scale mutagenesis along the entire 
 span of the tm segment , and measured their self - association in toxcat . 

each position was individually changed to a variety of large and small 
 hydrophobic amino acids .
the expectation is that the changes at interfacial 
 positions are more likely to perturb oligomerization than changes 
 at lipid exposed positions , as commonly observed ( for example ) .
the toxcat data is shown in figure s1 ( si ) and is schematically represented in figure 5a . to compute an overall position - dependent sensitivity to mutation
, 
 we applied a classification scheme for the variants phenotypes 
 using four categories ( dashed lines in figure 
 s1 ( si ) ) , labeled as  wt - like  ( > 80% of wild 
 type cat activity ) ,  mild  ( 5080% ) ,  severe  

position - based averaging reduces some of the natural 
 variability of the biological assay and the method has been reliable 
 in identifying the most sensitive positions at the helix 
mackenzie 
 plot  summarizing the effect of all mutations of adck3-tm measured 
 in toxcat .
the color coding of the gxxxg motifs in the sequence corresponds 
 to figure 1 .
a calculated average disruption 
 score for each position is displayed at the bottom of the scheme . 

( b ) the same average disruption plotted numerically ( 0 = as tw ; 3 
 = disruptive ) .
the mutagenesis reveals two positions that are essential 
 for self - association , g223 and g227 , which are the last two position 
 of adck3s gly - zipper ( red ) .
a majority of the variants had cat activity levels similar 
 or higher 
 compared to the wild type sequences ( figure 5b ) .
however , a number of variants showed dramatically reduced activity 
 in a position specific fashion . in particular , all variants of the 
 two c - terminal gly residues of the gly - zipper ( g223 and g227 ) have 
 the strongest disruptive phenotypes .
interestingly , the next most 
 sensitive positions are l220 and l224 , which are also predicted to 
 be interfacial in almost all catm models ( figure 3 ) .
conversely , the n - terminal positions of the zipper are 
 either mildly affected by mutation ( g219 ) or appear completely tolerant 
 ( a215 ) .
the off - frame 221-axxxg-225 ( magenta ) is also relatively 
 insensitive 
 to mutation .
substitution for a large leu at these two positions has 
 only a mild effect , and the ile variants are completely tolerated . 

this is consistent with the catm predictions , which do not identify 
 any model in which this motif is at the interface .
polar 
 residues can drive tm helix oligomerization through the formation 
 of hydrogen bonds , and have been found to be important for the association 
 of model peptides and of biological systems , including in the context of gasright motifs .
in addition , some polarity of position 216 appears to be relatively 
 conserved , as the main substitutions of n216 in a sequence alignment 
 ( figure 2c ) are gly , ser , gln and glu . however , 
 neither the computational nor the experimental analysis suggest that 
 n216 is important for self - association .
asn 216 can be mutated to 
 ala , leu or phe in toxcat without reduction of self - association .
catm 
 is in agreement with the experimental data , as it does not identify 
 any potential strong polar interaction ( i.e. , n 
ho 
 hydrogen bonds ) involving the side chain of n216 , although the side 
 chain carbonyl oxygen ( o1 ) acts as a ch bond 
 acceptor in most models .
overall the data indicates that the 
 interface of the adck3-tm oligomer 
 is mediated by gly - zipper motif and , in particular , by the c - terminal 
 side of this interaction motif . in order to identify the structure most consistent 
 with the toxcat data
, we performed a mutational analysis of the five 
 models generated by catm . using a protocol developed previously to 
 analyze similar mutational data
, we created 
 in silico the same set of variants that were tested experimentally , 
 and computed an analogous position - dependent  average disruption  

given that all catm structures interact through portions 
 of the extended gly - zipper , the computed patterns have similar periodicity 
 across all models , with disruption peaking at position g219 , g223 
 and/or g227 .
models 1 , 3 , and 5 ( figure 6a , c , e ) 
 show high sensitivity to mutation on the n - terminal side of the tm 
 domain , in disagreement with the experimental observations . in these 
 three models , mutations to g219
models 1 and 5 are 
 also very sensitive at positions a215 and n216 , which are completely 
 tolerant experimentally .
comparison 
 of the mutagenesis obtained in toxcat ( same as figure 5b ) with the computational mutagenesis performed on the five 
 catm models ( a  e ) .
the comparison suggests that model 2 is 
 the best fit to the experimental data , followed by model 4 .
( f ) a 
 linear combination of model 2 ( 60% ) and model 4 ( 40% ) produces an 
 excellent fit to the data , suggesting that the tm of adck3 may be 
 in equilibrium between at least two conformations in the toxcat system .
model 2 ( figure 6b ) and 
 model 4 ( figure 6d ) are in better agreement 
 with the experimental data and represent two possible structural solutions 
 for the adck3 tm dimer .
the disruption for both models peak at g223 
 and g227 , which are also the two most disruptive positions in toxcat . 

however , model 4 appears insensitive at position g219 ( which is mildly 
 sensitive experimentally ) and it is extremely disruptive at position 
 a228 ( which is insensitive experimentally ) .
in addition to being a 
 better match , model 2 also has lower energy , better packing and a 
 larger number of hydrogen bonds ( tables s2 and 
 s4 ( si ) ) .
therefore , model 2 appears to be the best structural 
 candidate for the adck3 tm dimer .
structural models 2 and 4 . comparison 
 of the structures of catm 
 models 2 and 4 for adck3 . from left to right , entire tm helix , detail 
 of the interface , and same conformation in full atom spheres .
model 
 2 has lower energy , a larger number of hydrogen bonds ( 12 in model 
 2 versus 4 in model 4 ) and more extended and complementary packing . in a recent analysis of known 
 gasright structures
, we 
 demonstrated that catm is capable of capturing alternative conformations 
 of biological importance .
therefore , 
 an additional possibility is that the tm domain of adck3 may be in 
 equilibrium between two or more structures .
we observed that a linear 
 combination of the mutagenesis profiles of the two models that best 
 fit the data , 60% of model 2 and 40% of model 4 , improves the fit 
 with the toxcat data , producing an excellent correspondence between 
 the two experiments ( figure 6f ) .
this interpolation 
 is not necessarily quantitative , but it suggests that a conformational 
 equilibrium would be compatible with the data . if such an equilibrium 
 occurs in the biological context , it would postulate that the tm domain 
 of adck3 may be a switchable element , a trait that could be important 
 for regulation or signaling , as observed in a number of other single - span 
 tm proteins . in this framework ,
the gly - zipper 
 would provide a dynamic interface for structural changes that could 
 potentially affect either the distance of the helical termini or the 
 relative rotation of the helices .
we 
 have presented a computational and experimental analysis of 
 the structural organization of the tm domain of the mitochondrial 
 kinase adck3 .
while more experiments are necessary to fully test catm , 
 the work provides a first practical demonstration of the applicability 
 of the program to the characterization of a tm dimer of unknown structure . 

it also confirms the ability of the algorithm ( which is based on optimization 
 of van der waals and ch hydrogen bonding ) to correctly 
 predict gasright motifs .
we have experimentally demonstrated 
 that the tm domain of adck3 
 self - associates in e. coli membranes . while the specific 
 oligomeric state could not be determined by toxcat
although gly - zipper motifs can be 
 involved in the formation of higher - oligomeric complexes , the good agreement between the experimental 
 and computational mutagenesis supports the homodimeric hypothesis . 

moreover , such oligomeric state is also consistent with a large body 
 of structural evidence , which shows that kinases frequently form dimeric 
 complexes ( for example ) , while higher oligomers are 
 rarely observed .
helix interaction interface was determined 
 and the mutagenesis identified a number of disruptive interfacial 
 mutations that will be useful for follow - up functional studies .
the 
 computational prediction of alternative models in which the helices 
 adopt a different crossing point along the gly - zipper interface raises 
 also the hypothesis that the tm domain of adck3 may possibly undergo 
 conformational changes .
indirectly , the work also provides important 
 insight about adck4 . 
 all the amino acids that participate at the dimerization interface 
 of adck3 are identical in adck4 .
the two positions that differ between 
 the two sequences ( f228l and a230 v , figure 2 ) are insensitive to variation when they are mutated individually 
 ( figure 5 ) .
the computational predictions obtained 
 for adck3 and adck4 are nearly identical , and it is thus expected 
 that both tm domains dimerize with the same structure . because the 
 two interfaces are compatible with each other , it also possible that 
 the tm domains could associate to drive formation of a heterodimeric 
 complex between adck3 and adck4 .
these hypotheses need to be investigated 
 in a biological context ; the present analysis provides the theoretical 
 foundation necessary for testing in vivo the role of these tm domains .
 interactions 
 between -helices within the hydrophobic environment 
 of lipid bilayers are integral to the folding and function of transmembrane 
 proteins ; however , the major forces that mediate these interactions 
 remain debated , and our ability to predict these interactions is still 
 largely untested . 
 we recently demonstrated that the frequent transmembrane 
 association motif gasright , the gxxxg - containing fold of 
 the glycophorin a dimer , is optimal for the formation of extended 
 networks of ch hydrogen bonds , supporting the hypothesis 
 that these bonds are major contributors to association . 
 we also found 
 that optimization of ch hydrogen bonding and interhelical 
 packing is sufficient to computationally predict the structure of 
 known gasright dimers at near atomic level . here , 
 we demonstrate 
 that this computational method can be used to characterize the structure 
 of a protein not previously known to dimerize , by predicting and validating 
 the transmembrane dimer of adck3 , a mitochondrial kinase . 
 adck3 is 
 involved in the biosynthesis of the redox active lipid , ubiquinone , 
 and human adck3 mutations cause a cerebellar ataxia associated with 
 ubiquinone deficiency , but the biochemical functions of adck3 remain 
 largely undefined . 
 our experimental analyses show that the transmembrane 
 helix of adck3 oligomerizes , with an interface based on an extended 
 gly - zipper motif , as predicted by our models . 
 the data provide strong 
 evidence for the hypothesis that optimization of ch 
 hydrogen bonding is an important factor in the association of transmembrane 
 helices . 
 this work also provides a structural foundation for investigating 
 the role of transmembrane association in regulating the biological 
 activity of adck3 . 
Introduction
Methods
Results and Discussion
Conclusions