the combination of structural genomics efforts and computational modeling has resulted in a large amount of 3d structure information for proteins . however , to a large degree , this structural information has not been translated into functional information . for example , understanding substrate specificity , catalysis or inhibition , is still largely dependent on biochemical and biophysical analysis of individual proteins . while protein structure in principle encodes this mechanistic information , reliable computational tools and approaches to establish a connection between structure and function are still lacking .
the molecular function of proteins is largely determined by their interaction with other molecules at binding sites on the protein surface .
thus , localization and characterization of a ligand binding site can contribute to functional annotation of a protein ; it can guide mutational experiments , and be useful in predicting or verifying interactions .
the identification of ligand binding sites can also be an important part of the drug discovery process . knowing the location of binding sites facilitates virtual screening for hits , lead optimization and identification of features that influence the selectivity of binding .
hence , several methods have been developed for the identification of binding sites from protein structures ( 16 ) and sequences ( 710 ) .
the structure - based methods recognize geometrical features , such as clefts , or energetic features that describe the molecular interaction properties of the protein surface . in general ,
structure - based methods can be seen as complementary to sequence - based methods that exploit evolutionary information . here
, we describe the sitehound - web server for identification of ligand binding sites in protein structures .
it uses an energy - based approach to identify regions with high potential for interaction with ligands .
a unique feature of sitehound - web is that it implements the use of different probes to characterize a protein structure , which enables not only the identification of different types of binding sites , but also a preliminary description of its interaction properties .
the sitehound algorithm identifies potential ligand binding sites by recognizing regions characterized by favorable non - bonded interactions with a chemical probe ( 6 ) . depending on the nature of the probe , different types of binding sites
currently , a  carbon probe and a  phosphate probe are available for the identification of binding sites for drug - like molecules , and ligands containing phosphate groups , respectively .
affinity maps ( also called molecular interaction fields ) describing the interaction of the probe and the protein on a regular 3d lattice are calculated using either the autogrid program ( 11 ) for the carbon probe , or the easymifs program ( d. ghersi and r. sanchez , manuscript submitted for publication ) for the phosphate probe .
the remaining points are clustered according to their spatial proximity using an agglomerative hierarchical clustering algorithm .
the final output is a list of  interaction energy clusters corresponding to putative binding sites , which are ranked by total interaction energy ( tie ) ( the sum of the energy values of all the points that belong to the same cluster ) . a test study carried out on 77 experimentally determined protein structures , corresponding to known protein 
ligand complexes , showed that the correct binding site was among the top three sitehound clusters in 95% of the cases ( 6 ) .
sitehound - web ( http://sitehound.sanchezlab.org ) was implemented using a python - cgi and javascript based platform .
wrappers integrate programs modeller ( 12 ) , autogrid ( 11 ) , easymifs ( d. ghersi and r. sanchez , manuscript submitted for publication ) , and sitehound ( 6 ) , resulting in a completely automated identification of ligand binding sites from a standard pdb file .
the processed pdb file is then passed to either autogrid or easymifs , depending on the user - selected probe .
the output is displayed using html pages including an interactive 3d representation of the protein structure and the putative binding sites using the jmol java applet ( http://www.jmol.org ) .
sitehound - web requires a pdb file as input and the specification of a probe and clustering algorithm for the calculation .
the input pdb file can either be uploaded or a pdb code can be specified . when specifying a pdb code the corresponding file is copied from the pdb database .
the pdb file does not need to be preprocessed ( e.g. removal of ligands ) since the server does this automatically .
two types of probes are currently available : a carbon probe for the identification of binding sites for molecules that interact mainly through van der waals contacts ; and a phosphate probe which is used to identify sites that bind to phosphorylated ligands . the carbon probe has been validated mainly with drug - like molecules ( 6 ) and the phosphate probe with phosphopeptides , phosphosugars , and atp ( d. ghersi and r. sanchez , manuscript in preparation ) .
the clustering algorithm determines the way in which sitehound combines individual affinity map points into clusters corresponding to putative binding sites .
the average - linkage clustering tends to result in relatively spherical clusters and is the default for both probes .
while only the use of average - linkage clustering has been tested extensively in sitehound , the single - linkage clustering algorithm is provided as an alternative to be used with the carbon probe for the identification of larger elongated binding sites , like those of peptides .
once a request has been submitted , the calculation proceeds unless a multiple chain pdb file has been uploaded or selected . in this case
, the server will provide the option to select one or more chains from the pdb file to be included in the calculation .
after chain selection the calculation proceeds . for a medium - sized protein ( 150 residues ) , a typical calculation takes 1  min .
however , running time also depends on the shape of the protein , with elongated proteins taking longer than spherical ones .
the output of sitehound - web has two components : an interactive web screen displaying a summary of results with a 3d representation of the putative binding sites on the protein structure ; and downloadable files for offline analysis .
a  cluster data table ( figure 1a ) displays the top 10 ranking interaction energy clusters ( i.e. putative binding sites ) .
this table shows the rank , tie , coordinates , and volume for each cluster .
the color of the rank corresponds to the color of the cluster in the 3d display .
the tie , which is used to rank the clusters , is an indication of the strength of the clusters .
significant clusters usually have ties that stand out against the background of weaker clusters ( see clusters 1 and 2 in figure 1a ; and cluster 1 in figure 2a ) .
the cluster coordinates correspond to the x , y and z coordinates of the center of each cluster .
this can be used , for example , to set up a docking box centered around a putative binding site ( 6 ) .
finally , the volume of the cluster in  is displayed in the last column .
a 3d interactive view of the protein structure and the clusters ( figure 1b ) is provided using the jmol molecular viewer .
this view interacts with the  cluster selection panel ( figure 1c ) , which can be used to toggle the display of any of the top 10 clusters on and off .
a  cluster details panel ( figure 1d ) provides a list of protein residues in the vicinity of a selected cluster . clicking on its corresponding rank in the cluster data table changes the selected cluster .
finally , the  download data panel ( figure 1e ) provides links to various data files .
the  cluster data file provides the same information as the cluster data table , but for all identified clusters .
the dx file stores cluster data in the dx format which is useful for display in programs such as pymol ( http://www.pymol.org ) and chimera ( 13 ) .
the cluster pdb file contains the coordinates of the cluster points in pdb format ; it can be used to display the clusters in most molecular viewers ( figure 3 ) and is the file used internally by sitehound - web to display the clusters using jmol .
it can be used with the offline version of sitehound ( d. ghersi and r. sanchez , manuscript submitted for publication ) to explore different parameters for cluster analysis . 

the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the carbon probe and the average - linkage clustering algorithm is shown .
( a ) the  cluster data table summarizes the information for the top 10 clusters ranked by total interaction energy .
the cluster number indicates the rank of the cluster with the colors corresponding to the coloring of the cluster in the structure display and cluster selection windows .
two clusters ( circled with the dotted line ) stand out has having significantly more favorable interaction energy than the rest .
the coordinates for the center of the cluster and the cluster volume are also displayed .
( b ) the structure display window provides a 3d view of the clusters in the context of the protein structure using the jmol java applet ( http://www.jmol.org ) .
cluster selection panel allows toggling the display of individual clusters on or off . by default
( d ) the  cluster details panel displays all residues in contact with the cluster selected in the cluster data window .
( e ) the  download data panel provides links to various data files for offline analysis ( see text for a description of each file ) . 

the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the phosphate probe and the average - linkage clustering algorithm is shown . only the structure display ( a ) and
cluster 1 ( circled ) stands out as having significantly more favorable interaction energy with the phosphate probe than the rest of the clusters .
the position of cluster 1 is intermediate between the two most favorable carbon probe clusters ( figures 1 and 3 ) . 
 figure 3.combining sitehound - web outputs to describe the adenylate kinase binding site .
( a ) ribbon diagram of the yeast adenylate kinase structure showing the top ranking clusters from figures 1 and 2 as solid surfaces : phosphate probe cluster ( red ) and carbon probe clusters ( green ) .
( b ) sitehound - web clusters superposed on the structure of the ap5a ( bis(adenosine)-5-pentaphosphate ) inhibitor of adenylate kinase ( 14 ) .
the phosphate probe correctly identifies the pathway of phosphoryl transfer , and the carbon probe correctly identifies the adenosine binding regions .
the figure was prepared using the  cluster pdb file downloadable output files from sitehound - web examples shown in figures 1 and 2 , and the pymol molecular graphics program ( http://www.pymol.org ) .
the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the carbon probe and the average - linkage clustering algorithm is shown .
( a ) the  cluster data table summarizes the information for the top 10 clusters ranked by total interaction energy .
the cluster number indicates the rank of the cluster with the colors corresponding to the coloring of the cluster in the structure display and cluster selection windows .
two clusters ( circled with the dotted line ) stand out has having significantly more favorable interaction energy than the rest .
the coordinates for the center of the cluster and the cluster volume are also displayed .
( b ) the structure display window provides a 3d view of the clusters in the context of the protein structure using the jmol java applet ( http://www.jmol.org ) .
cluster selection panel allows toggling the display of individual clusters on or off . by default
( d ) the  cluster details panel displays all residues in contact with the cluster selected in the cluster data window .
( e ) the  download data panel provides links to various data files for offline analysis ( see text for a description of each file ) .
the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the phosphate probe and the average - linkage clustering algorithm is shown . only the structure display ( a ) and cluster data ( b ) panels are shown .
cluster 1 ( circled ) stands out as having significantly more favorable interaction energy with the phosphate probe than the rest of the clusters .
the position of cluster 1 is intermediate between the two most favorable carbon probe clusters ( figures 1 and 3 ) .
( a ) ribbon diagram of the yeast adenylate kinase structure showing the top ranking clusters from figures 1 and 2 as solid surfaces : phosphate probe cluster ( red ) and carbon probe clusters ( green ) .
( b ) sitehound - web clusters superposed on the structure of the ap5a ( bis(adenosine)-5-pentaphosphate ) inhibitor of adenylate kinase ( 14 ) .
the phosphate probe correctly identifies the pathway of phosphoryl transfer , and the carbon probe correctly identifies the adenosine binding regions .
the figure was prepared using the  cluster pdb file downloadable output files from sitehound - web examples shown in figures 1 and 2 , and the pymol molecular graphics program ( http://www.pymol.org ) .
the sitehound algorithm identifies potential ligand binding sites by recognizing regions characterized by favorable non - bonded interactions with a chemical probe ( 6 ) . depending on the nature of the probe , different types of binding sites
currently , a  carbon probe and a  phosphate probe are available for the identification of binding sites for drug - like molecules , and ligands containing phosphate groups , respectively .
affinity maps ( also called molecular interaction fields ) describing the interaction of the probe and the protein on a regular 3d lattice are calculated using either the autogrid program ( 11 ) for the carbon probe , or the easymifs program ( d. ghersi and r. sanchez , manuscript submitted for publication ) for the phosphate probe .
the remaining points are clustered according to their spatial proximity using an agglomerative hierarchical clustering algorithm .
the final output is a list of  interaction energy clusters corresponding to putative binding sites , which are ranked by total interaction energy ( tie ) ( the sum of the energy values of all the points that belong to the same cluster ) . a test study carried out on 77 experimentally determined protein structures , corresponding to known protein 
ligand complexes , showed that the correct binding site was among the top three sitehound clusters in 95% of the cases ( 6 ) .
sitehound - web ( http://sitehound.sanchezlab.org ) was implemented using a python - cgi and javascript based platform .
a series of python  wrappers integrate programs modeller ( 12 ) , autogrid ( 11 ) , easymifs ( d. ghersi and r. sanchez , manuscript submitted for publication ) , and sitehound ( 6 ) , resulting in a completely automated identification of ligand binding sites from a standard pdb file .
the processed pdb file is then passed to either autogrid or easymifs , depending on the user - selected probe . the resulting affinity map is then passed to sitehound .
the output is displayed using html pages including an interactive 3d representation of the protein structure and the putative binding sites using the jmol java applet ( http://www.jmol.org ) .
sitehound - web requires a pdb file as input and the specification of a probe and clustering algorithm for the calculation .
the input pdb file can either be uploaded or a pdb code can be specified . when specifying a pdb code the corresponding file is copied from the pdb database .
the pdb file does not need to be preprocessed ( e.g. removal of ligands ) since the server does this automatically .
two types of probes are currently available : a carbon probe for the identification of binding sites for molecules that interact mainly through van der waals contacts ; and a phosphate probe which is used to identify sites that bind to phosphorylated ligands . the carbon probe has been validated mainly with drug - like molecules ( 6 ) and the phosphate probe with phosphopeptides , phosphosugars , and atp ( d. ghersi and r. sanchez , manuscript in preparation ) .
the clustering algorithm determines the way in which sitehound combines individual affinity map points into clusters corresponding to putative binding sites .
the average - linkage clustering tends to result in relatively spherical clusters and is the default for both probes .
while only the use of average - linkage clustering has been tested extensively in sitehound , the single - linkage clustering algorithm is provided as an alternative to be used with the carbon probe for the identification of larger elongated binding sites , like those of peptides .
once a request has been submitted , the calculation proceeds unless a multiple chain pdb file has been uploaded or selected . in this case
, the server will provide the option to select one or more chains from the pdb file to be included in the calculation .
after chain selection the calculation proceeds . for a medium - sized protein ( 150 residues ) , a typical calculation takes 1  min .
however , running time also depends on the shape of the protein , with elongated proteins taking longer than spherical ones .
the output of sitehound - web has two components : an interactive web screen displaying a summary of results with a 3d representation of the putative binding sites on the protein structure ; and downloadable files for offline analysis .
a  cluster data table ( figure 1a ) displays the top 10 ranking interaction energy clusters ( i.e. putative binding sites ) .
this table shows the rank , tie , coordinates , and volume for each cluster .
the color of the rank corresponds to the color of the cluster in the 3d display .
the tie , which is used to rank the clusters , is an indication of the strength of the clusters .
significant clusters usually have ties that stand out against the background of weaker clusters ( see clusters 1 and 2 in figure 1a ; and cluster 1 in figure 2a ) .
the cluster coordinates correspond to the x , y and z coordinates of the center of each cluster .
this can be used , for example , to set up a docking box centered around a putative binding site ( 6 ) .
finally , the volume of the cluster in  is displayed in the last column .
a 3d interactive view of the protein structure and the clusters ( figure 1b ) is provided using the jmol molecular viewer .
this view interacts with the  cluster selection panel ( figure 1c ) , which can be used to toggle the display of any of the top 10 clusters on and off .
the coloring of the clusters corresponds to their rank in the cluster data table . a  cluster details panel ( figure 1d ) provides a list of protein residues in the vicinity of a selected cluster . clicking on its corresponding rank in the cluster data table changes the selected cluster .
finally , the  download data panel ( figure 1e ) provides links to various data files .
cluster data file provides the same information as the cluster data table , but for all identified clusters .
the dx file stores cluster data in the dx format which is useful for display in programs such as pymol ( http://www.pymol.org ) and chimera ( 13 ) .
the cluster pdb file contains the coordinates of the cluster points in pdb format ; it can be used to display the clusters in most molecular viewers ( figure 3 ) and is the file used internally by sitehound - web to display the clusters using jmol .
it can be used with the offline version of sitehound ( d. ghersi and r. sanchez , manuscript submitted for publication ) to explore different parameters for cluster analysis . 

the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the carbon probe and the average - linkage clustering algorithm is shown .
( a ) the  cluster data table summarizes the information for the top 10 clusters ranked by total interaction energy .
the cluster number indicates the rank of the cluster with the colors corresponding to the coloring of the cluster in the structure display and cluster selection windows .
two clusters ( circled with the dotted line ) stand out has having significantly more favorable interaction energy than the rest .
the coordinates for the center of the cluster and the cluster volume are also displayed .
( b ) the structure display window provides a 3d view of the clusters in the context of the protein structure using the jmol java applet ( http://www.jmol.org ) .
cluster selection panel allows toggling the display of individual clusters on or off . by default
( d ) the  cluster details panel displays all residues in contact with the cluster selected in the cluster data window .
data panel provides links to various data files for offline analysis ( see text for a description of each file ) . 

the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the phosphate probe and the average - linkage clustering algorithm is shown . only the structure display ( a ) and cluster data ( b ) panels are shown .
cluster 1 ( circled ) stands out as having significantly more favorable interaction energy with the phosphate probe than the rest of the clusters .
the position of cluster 1 is intermediate between the two most favorable carbon probe clusters ( figures 1 and 3 ) . 
 figure 3.combining sitehound - web outputs to describe the adenylate kinase binding site .
( a ) ribbon diagram of the yeast adenylate kinase structure showing the top ranking clusters from figures 1 and 2 as solid surfaces : phosphate probe cluster ( red ) and carbon probe clusters ( green ) .
( b ) sitehound - web clusters superposed on the structure of the ap5a ( bis(adenosine)-5-pentaphosphate ) inhibitor of adenylate kinase ( 14 ) .
the phosphate probe correctly identifies the pathway of phosphoryl transfer , and the carbon probe correctly identifies the adenosine binding regions .
the figure was prepared using the  cluster pdb file downloadable output files from sitehound - web examples shown in figures 1 and 2 , and the pymol molecular graphics program ( http://www.pymol.org ) .
the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the carbon probe and the average - linkage clustering algorithm is shown .
( a ) the  cluster data table summarizes the information for the top 10 clusters ranked by total interaction energy .
the cluster number indicates the rank of the cluster with the colors corresponding to the coloring of the cluster in the structure display and cluster selection windows .
two clusters ( circled with the dotted line ) stand out has having significantly more favorable interaction energy than the rest .
the coordinates for the center of the cluster and the cluster volume are also displayed .
( b ) the structure display window provides a 3d view of the clusters in the context of the protein structure using the jmol java applet ( http://www.jmol.org ) .
cluster selection panel allows toggling the display of individual clusters on or off . by default
( d ) the  cluster details panel displays all residues in contact with the cluster selected in the cluster data window .
( e ) the  download data panel provides links to various data files for offline analysis ( see text for a description of each file ) .
the output for yeast adenylate kinase ( 14 ) ( pdb code 1aky ) processed with the phosphate probe and the average - linkage clustering algorithm is shown . only the structure display ( a ) and cluster data ( b ) panels are shown .
cluster 1 ( circled ) stands out as having significantly more favorable interaction energy with the phosphate probe than the rest of the clusters .
the position of cluster 1 is intermediate between the two most favorable carbon probe clusters ( figures 1 and 3 ) . combining sitehound - web outputs to describe the adenylate kinase binding site .
( a ) ribbon diagram of the yeast adenylate kinase structure showing the top ranking clusters from figures 1 and 2 as solid surfaces : phosphate probe cluster ( red ) and carbon probe clusters ( green ) .
( b ) sitehound - web clusters superposed on the structure of the ap5a ( bis(adenosine)-5-pentaphosphate ) inhibitor of adenylate kinase ( 14 ) .
the phosphate probe correctly identifies the pathway of phosphoryl transfer , and the carbon probe correctly identifies the adenosine binding regions .
the figure was prepared using the  cluster pdb file downloadable output files from sitehound - web examples shown in figures 1 and 2 , and the pymol molecular graphics program ( http://www.pymol.org ) .
ligand binding site identification is an important tool in structural biology because it can bridge the structure - function gap in a homology - independent way .
sitehound - web is a ligand binding site identification server that can provide information about the location and binding preference of sites in protein structures .
it has a simple interface that only requires the user to select a protein structure and two options ( probe and clustering algorithm ) .
a unique feature of sitehound - web is its ability to identify different types of binding sites depending on the probe used for calculation .
future development of sitehound will include the addition of more probes for characterization of a more diverse set of sites . because the method requires only the structure of the protein as input
it can be used to complement sequence - based methods for identification of functionally important residues , which rely on evolutionary information .
we expect sitehound - web to be especially useful in the context of structural annotation , and docking applications in which the binding site is unknown .
while binding site identification methods can help in locating and characterizing the regions of the protein to which a ligand may bind , they can not guarantee that a given site will or will not bind a ligand .
this is a problem that is better addressed by techniques such as virtual screening that can be carried out on the putative binding sites .
national science foundation ( mcb 0517352 ) ; the national institutes of health ( gm081713 ) . funding for open access charge : national institutes of health ( gm081713 ) .
 sitehound - web ( http://sitehound.sanchezlab.org ) is a binding - site identification server powered by the sitehound program . given a protein structure in pdb format sitehound - web 
 will identify regions of the protein characterized by favorable interactions with a probe molecule . 
 these regions correspond to putative ligand binding sites . depending on the probe used in the calculation 
 , sites with preference for different ligands will be identified . 
 currently , a carbon probe for identification of binding sites for drug - like molecules , and a phosphate probe for phosphorylated ligands ( atp , phoshopeptides , etc . ) have been implemented . 
 sitehound - web will display the results in html pages including an interactive 3d representation of the protein structure and the putative sites using the jmol java applet . 
 various downloadable data files are also provided for offline data analysis . 
INTRODUCTION
METHODS
The SITEHOUND algorithm
Server implementation
SITEHOUND-web input
SITEHOUND-web output
CONCLUSIONS
FUNDING