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The FTMap family of web servers for determining and characterizing ligand-binding hot spots of proteins - PubMed

The FTMap family of web servers for determining and characterizing ligand-binding hot spots of proteins

Dima Kozakov et al. Nat Protoc. 2015 May.

Abstract

FTMap is a computational mapping server that identifies binding hot spots of macromolecules-i.e., regions of the surface with major contributions to the ligand-binding free energy. To use FTMap, users submit a protein, DNA or RNA structure in PDB (Protein Data Bank) format. FTMap samples billions of positions of small organic molecules used as probes, and it scores the probe poses using a detailed energy expression. Regions that bind clusters of multiple probe types identify the binding hot spots in good agreement with experimental data. FTMap serves as the basis for other servers, namely FTSite, which is used to predict ligand-binding sites, FTFlex, which is used to account for side chain flexibility, FTMap/param, used to parameterize additional probes and FTDyn, for mapping ensembles of protein structures. Applications include determining the druggability of proteins, identifying ligand moieties that are most important for binding, finding the most bound-like conformation in ensembles of unliganded protein structures and providing input for fragment-based drug design. FTMap is more accurate than classical mapping methods such as GRID and MCSS, and it is much faster than the more-recent approaches to protein mapping based on mixed molecular dynamics. By using 16 probe molecules, the FTMap server finds the hot spots of an average-size protein in <1 h. As FTFlex performs mapping for all low-energy conformers of side chains in the binding site, its completion time is proportionately longer.

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Conflict of interest statement

Competing financial interests

The authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/nprot/index.html.

Figures

FIGURE 1
FIGURE 1

Principles and tools of the FTMap algorithm. (a) Visualization of the FTMap process. In Step 1, probe molecules are docked. They are then energy minimized to find the most favorable positions (Step 2) and each probe type is clustered to yield probe clusters. These probe clusters are then clustered on the basis of their average free energy in Step 3 to yield consensus clusters, which defines the consensus sites (CSs). The CS with the largest number of probe clusters is the main hot spot; all other CSs are secondary hot spots. (b) The 16 small organic probe molecules that are used by FTMap. These probes vary in size, shape, and polarity. (c) An example of a consensus cluster in a protein pocket. The probes molecules depicted in this case are representatives of their probe clusters. The figure shows the binding pose of the lowest energy structure from each of the 16 probe clusters that define the hot spot. The color codes represent different atoms, carbon cyan, oxygen red, nitrogen blue, and hydrogen white.

FIGURE 2
FIGURE 2

Flowchart of the FTMap algorithm. Users upload a PDB to the server manually or using pdb.org. FTMap checks the structure, removes bound ligands and water molecules, and adds any missing atoms, including polar hydrogens. After calculation of the Poisson-Boltzman potential, positions for the first probe molecule are sampled using rigid body docking. This step uses the FFT correlation approach and a detailed energy expression, including terms for the van der Waals energy, the electrostatic interaction energy, a cavity term describing the hydrophobic contributions of the cavity and a knowledge-based pairwise potential. The 2000 best probe poses are retained and minimized using the CHARMM potential, which includes solvation. The probes are then clustered starting with the lowest energy structure and using a 3Å clustering radius. Clusters with less than 10 members are excluded from consideration. The clusters are ranked on the basis of their Boltzmann averaged energies and the six lowest energy clusters are retained. This step is repeated for all remaining probe molecules. When all 16 probe molecule types have been sampled and clustered, the probe clusters themselves are clustered. The probe cluster with the maximum number of neighbors within 4 Å is selected as the top consensus site (CS000) and all probe clusters within 4 Å of this CS are included. This procedure is repeated until all probe clusters have been assigned to a CS. CSs are ranked on the basis of the number of their probe clusters.

FIGURE 3
FIGURE 3

Flowchart of the FTFlex algorithm. Stage 1 is the identification of the hot spots using the FTMap algorithm. After Stage 1 is completed, the program stops, and the user can select the ligand binding region, or other region of interest, by specifying the consensus sites that define this region. After this selection the server proceeds to Stage 2, considering the residues that are within 5Å of the selected hot spots and satisfy hydrophobicity and cavity size restrictions. In Stage 2 possible rotamers are determined for each of the selected residue, inserted back into the initial PDB structure, and the resulting structures are mapped again using FTMap. After all rotamers have been tested, the rotamers that yield the most improvement are substituted back into the initial PDB file to yield a modified structure, which is mapped and the results are returned to the user.

FIGURE 4
FIGURE 4

Screen image of FTMap results for PDB ID 2ren (apo structure of renin). Five different output files are available for download; descriptions can be found in Box 3. The user can view the mapping results using the PyMol plugin, or by downloading either the PDB file containing the protein and probe coordinate or a PyMol session. The target protein is shown as a green cartoon, and the probes representing each cluster at the consensus sites are shown in various colors as sticks. Clicking on the image will activate JSmol, and the image can be manipulated (e.g., rotated and translated) using the JSmol tools. JSmol also provides checkboxes along the bottom of the picture of the protein to select/deselect any of the consensus clusters. The two bar graphs at the bottom of the page show the percent of nonbonded and hydrogen bond interactions, respectively, between the probes and the protein for each residue along the protein sequence. You can download these results as tab separated files with exact residue contact counts by clicking the link located directly beneath each respective graph.

FIGURE 5
FIGURE 5

Viewing FTMap results for PDB ID 2ren (apo structure of renin) using PyMOL. (a) Opening the PyMOL session provided by the server. In addition to the protein and the representative probes at the consensus sites, PyMOL lists consensus clusters on the right hand side of the screen in order of probe cluster ranking. The format of the consensus clusters is “crossclusters.xxx.yyy” where xxx is the ranking of the consensus site, starting at 000, and yyy is the number of probe clusters. (b) Demonstrating the value of looking at the FTMap results using PyMOL: The inhibitor Aliskiren (magenta sticks) from the inhibitor-bound renin structure 2v0z is superimposed onto the hot spots spots calculated for the renin apo structure, which shows that the entire inhibitor binding site is covered by hot spots (For better viewing the protein is not shown).

FIGURE 6
FIGURE 6

Screen capture of FTSite results for human lymphocyte kinase (Lck, PDB ID 3lck). A bound inhibitor from the structure with PDB ID 1qpe is superimposed for reference, shown in white ball-and-stick representation. The figure on the left shows the top prediction of the ligand binding site, named Site 1, using mesh representation for the cluster of probe molecules found at this site. The output page also lists the residues that are within 5 Å of the binding site found. The plot reveals that the binding site identified covers only about half of the inhibitor. While site 2 extends the binding pocket in a direction that does not interact with the this particular inhibitor, adding Site 3 to Site 1 covers the entire region of inhibitor binding (see figure on the right). The residues interacting with Site 3 are also listed.

FIGURE 7
FIGURE 7

Transition from Stage 1 to Stage 2 in FTFlex: selection of consensus clusters around which low energy side chain conformers will be explored in repeated mapping calculations. The figure shows the output page from Stage 1 of FTFlex applied to Chain A of the apo structure of the cyclin-dependent kinase 2 (CDK2, PDB ID 1pw2). Consensus sites 002 and 004 were selected by clicking on the appropriate buttons. Notice the small “Submit” button for starting Stage 2 of FTFlex.

FIGURE 8
FIGURE 8

Screenshot of the PyMOL session from the application of FTMap/param to the unbound structure of thrombin (PDB ID: 1hxf) with the user-defined probe molecule 1-(3-chlorophenyl)methanamine, a thrombin inhibitor. We show the lowest energy pose for conformer 1 of this additional probe (yellow sticks), overlapping with the largest consensus site (cyan, 30 probe clusters). The 1-(3-chlorophenyl)methanamine molecule bound to thrombin (magenta) from the structure with PDB ID 2c8z is superimposed for reference.

FIGURE 9
FIGURE 9

Screenshots of the result page when applying FTDyn to map an ensemble of 24 structures of the MDM2 protein (PDB ID 1z1m), determined by Nuclear Magnetic Resonace (NMR). (a) The page shows the first structure of the ensemble in cartoon representation, but the color coding is based on the number of probe-protein contacts averaged over the entire ensemble. Residues are colored from blue to red based on contact frequency. Clicking on the picture of the molecule activates JSmol, and thus the structure can be rotated and translated. (b) The two bar graphs on the page show the percent of nonbonded and hydrogen bond interactions, respectively, averaged over all structures. Below the graphs the table can be used to view or download mapping results for all structures together, or any of the structures of the ensemble individually. Clicking on the Load button under the Map tag will activate JSmol for the visualization of the selected structure. The PDB file of the mapping results and the lists of nonbonded and hydrogen bond interactions can also be downloaded for each structure.

FIGURE 10
FIGURE 10

Mapping of the 24 MDM2 structures obtained by NMR using FTDyn. (a) Screenshot of the PyMOL session shows the ensemble, with residues color-coded from blue to red based on contact frequency. (b) Comparing the distribution of nonbonded probe-protein contacts for residues of MDM2, based on the mapping of model 9 in the ensemble (blue bars), to the distribution of nonbonded ligand-protein contacts observed in the complex of MDM2 with piperidinone, a small molecular inhibitor of the MDM2-p53 interaction (PDB ID 2lzg), shown as red bars. Horizontal axes list residues of MDM2 from Glu25 to Tyr104 (unstructured regions were removed before mapping analysis). The vertical axis shows the fraction of atom-atom interactions that each protein residue makes with probe or ligand atoms.

FIGURE 11
FIGURE 11

Comparison of FTMap results to experimental data. (a) Probe binding to Ribonuclease A (RNase A), observed by the Multiple Solvent Crystal Structures (MSCS) methods using X-ray crystallography. In all cases, the CSs are shown as colored sticks and RNase A is shown as a tan surface. The sites identified using MSCS are circled and labeled as B1, B2, and P1. (b) Consensus sites for the mapping of the unbound structure of RNase using PDB ID 2e3w. The CSs are as follows: CS000 (20 probe clusters, cyan), CS001 (16 probe clusters, magenta), CS002 (13 probe clusters, yellow), CS003 (13 probe clusters, salmon), and CS004 (11 probe clusters, white). Although CS002 is not seen in the MSCS experiments, the existence of a hot spot at that location has been demonstrated by alanine scanning data (see Anticipated results).

FIGURE 12
FIGURE 12

Consensus sites identified using FTMap for thrombin (PDB ID 1ths) are shown in line representation, overlapping with fragments and a high affinity ligand shown as sticks. Mapping identifies 4 consensus sites (left). Of these sites, CS000 (cyan) has 25 probe clusters, CS002 (yellow) has 13 probe clusters, CS004 (white) has 7 probe clusters, and CS005 (periwinkle) has 7 probe clusters. These consensus sites overlap with two known fragments (middle), Fragment 1 (fushia sticks, PDB ID 2c90) and Fragment 2 (pale green sticks, PDB ID 2c93). Joining the two fragments to take full advantage of all hot spots yields an inhibitor (PDB ID 2c8w) with an IC50 value of 4 nM, shown in teal sticks overlaid with the consensus sites.

FIGURE 13
FIGURE 13

Flexible mapping the apo structure of the cyclin-dependent kinase 2 (CDK2, PDB ID 1pw2) using FTFlex. (a) Results from Stage 1, i.e., without adjusting the side chains in the X-ray structure. The figure shows CS002 (green, 13 probe clusters) and CS004 (magenta, 10 probe clusters) in the ligand binding site. An inhibitor bound to CDK2 (from the structure with PDB ID 1ke5) is superimposed for reference (grey sticks). We also show two “moving” side chains, Lys33 and Lys89, that with their conformers in the apo structure protrude into the ligand binding site and substantially reduce its volume (brown sticks). In the inhibitor-bound structure (PDB ID 1ke5) the two side chains move out of the pocket, and thus do not interfere with ligand binding (also shown as grey sticks). (b) Results from Stage 2 of FTFlex. Both Lys33 and Lys89, shown as brown sticks, have largely moved out from the ligand binding site. The Lys33 side chain is close to the conformer in the inhibitor-bound structure 1ke5. Although this is not the case for Lys89, the side chain moves farther from the pocket, and thus does not interfere with probe binding. As A result, the ligand binding site now includes the two most populated consensus sites, CS000 (green, 18 probe clusters) and CS001 (magenta, 14 probe clusters).

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