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1.  Fragment-based identification of druggable ‘hot spots’ of proteins using Fourier domain correlation techniques 
Bioinformatics  2009;25(5):621-627.
Motivation: The binding sites of proteins generally contain smaller regions that provide major contributions to the binding free energy and hence are the prime targets in drug design. Screening libraries of fragment-sized compounds by NMR or X-ray crystallography demonstrates that such ‘hot spot’ regions bind a large variety of small organic molecules, and that a relatively high ‘hit rate’ is predictive of target sites that are likely to bind drug-like ligands with high affinity. Our goal is to determine the ‘hot spots’ computationally rather than experimentally.
Results: We have developed the FTMAP algorithm that performs global search of the entire protein surface for regions that bind a number of small organic probe molecules. The search is based on the extremely efficient fast Fourier transform (FFT) correlation approach which can sample billions of probe positions on dense translational and rotational grids, but can use only sums of correlation functions for scoring and hence is generally restricted to very simple energy expressions. The novelty of FTMAP is that we were able to incorporate and represent on grids a detailed energy expression, resulting in a very accurate identification of low-energy probe clusters. Overlapping clusters of different probes are defined as consensus sites (CSs). We show that the largest CS is generally located at the most important subsite of the protein binding site, and the nearby smaller CSs identify other important subsites. Mapping results are presented for elastase whose structure has been solved in aqueous solutions of eight organic solvents, and we show that FTMAP provides very similar information. The second application is to renin, a long-standing pharmaceutical target for the treatment of hypertension, and we show that the major CSs trace out the shape of the first approved renin inhibitor, aliskiren.
Availability: FTMAP is available as a server at http://ftmap.bu.edu/.
Contact: vajda@bu.edu
Supplementary information: Supplementary Material is available at Bioinformatics online.
doi:10.1093/bioinformatics/btp036
PMCID: PMC2647826  PMID: 19176554
2.  FTMAP: extended protein mapping with user-selected probe molecules 
Nucleic Acids Research  2012;40(Web Server issue):W271-W275.
Binding hot spots, protein sites with high-binding affinity, can be identified using X-ray crystallography or NMR by screening libraries of small organic molecules that tend to cluster at such regions. FTMAP, a direct computational analog of the experimental screening approaches, globally samples the surface of a target protein using small organic molecules as probes, finds favorable positions, clusters the conformations and ranks the clusters on the basis of the average energy. The regions that bind several probe clusters predict the binding hot spots, in good agreement with experimental results. Small molecules discovered by fragment-based approaches to drug design also bind at the hot spot regions. To identify such molecules and their most likely bound positions, we extend the functionality of FTMAP (http://ftmap.bu.edu/param) to accept any small molecule as an additional probe. In its updated form, FTMAP identifies the hot spots based on a standard set of probes, and for each additional probe shows representative structures of nearby low energy clusters. This approach helps to predict bound poses of the user-selected molecules, detects if a compound is not likely to bind in the hot spot region, and provides input for the design of larger ligands.
doi:10.1093/nar/gks441
PMCID: PMC3394268  PMID: 22589414
3.  Hot spot analysis for driving the development of hits into leads in fragment based drug discovery 
Fragment based drug design (FBDD) starts with finding fragment-sized compounds that are highly ligand efficient and can serve as a core moiety for developing high affinity leads. Although the core-bound structure of a protein facilitates the construction of leads, effective design is far from straightforward. We show that protein mapping, a computational method developed to find binding hot spots and implemented as the FTMap server, provides information that complements the fragment screening results and can drive the evolution of core fragments into larger leads with a minimal loss or, in some cases, even a gain in ligand efficiency. The method places small molecular probes, the size of organic solvents, on a dense grid around the protein, and identifies the hot spots as consensus clusters formed by clusters of several probes. The hot spots are ranked based on the number of probe clusters, which predicts the binding propensity of the subsites and hence their importance for drug design. Accordingly, with a single exception the main hot spot identified by FTMap binds the core compound found by fragment screening. The most useful information is provided by the neighboring secondary hot spots, indicating the regions where the core can be extended to increase its affinity. To quantify this information, we calculate the density of probes from mapping, which describes the binding propensity at each point, and show that the change in the correlation between a ligand position and the probe density upon extending or repositioning the core moiety predicts the expected change in ligand efficiency.
doi:10.1021/ci200468p
PMCID: PMC3264775  PMID: 22145575
Protein mapping; protein docking; drug design; ligand efficiency; affinity prediction
4.  Analysis of protein binding sites by computational solvent mapping 
Summary
Computational solvent mapping globally samples the surface of target proteins using molecular probes – small molecules or functional groups – to identify potentially favorable binding positions. The method is based on X-ray and NMR screening studies showing that the binding sites of proteins also bind a large variety of fragment-sized molecules. We have developed the multi-stage mapping algorithm FTMap (available as a server at http://ftmap.bu.edu/) based on the fast Fourier transform (FFT) correlation approach. Identifying regions of low free energy rather than individual low energy conformations, FTMap reproduces the available experimental mapping results. Applications to a variety of proteins show that the probes always cluster in important subsites of the binding site, and the amino acid residues that interact with many probes also bind the specific ligands of the protein. The “consensus” sites at which a number of different probes cluster are likely to be “druggable” sites, capable of binding drug-size ligands with high affinity. Due to its sensitivity to conformational changes the method can also be used for comparing the binding sites in different structures of a protein.
doi:10.1007/978-1-61779-465-0_2
PMCID: PMC3526383  PMID: 22183527
Protein structure; protein-ligand interactions; binding site; binding hot spots; fragment-based ligand design; druggability; binding site comparison; docking
5.  Analysis of Binding Site Hot Spots on the Surface of Ras GTPase 
Journal of molecular biology  2011;413(4):773-789.
We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the “off” and “on” allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.
doi:10.1016/j.jmb.2011.09.011
PMCID: PMC3247908  PMID: 21945529
Ras isoforms; drug target; binding site hot spots; Ras dynamics; allosteric switch
6.  Detection of ligand binding hot spots on protein surfaces via fragment-based methods: Application to DJ-1 and Glucocerebrosidase 
Journal of computer-aided molecular design  2009;10.1007/s10822-009-9283-2.
The identification of hot spots, i.e. binding regions that contribute substantially to the free energy of ligand binding, is a critical step for structure-based drug design. Here we present the application of two fragment-based methods to the detection of hot spots for DJ-1 and glucocerebrosidase (GCase), targets for the development of therapeutics for Parkinson’s and Gaucher’s diseases respectively. While the structures of these two proteins are known, binding information is lacking. In this study we employ both the multiple solvent crystal structures (MSCS) method and the FTMap algorithm to identify regions suitable for the development of pharmacological chaperones for DJ-1 and GCase. Comparison of data derived via MSCS and FTMap also shows that FTMap, a computational method for the identification of fragment binding hot spots, is an accurate and robust alternative to the performance of expensive and difficult MSCS experiments.
doi:10.1007/s10822-009-9283-2
PMCID: PMC2889209  PMID: 19521672
fragment-based drug design; structure-based drug design; hot spot identification; DJ-1; glucocerebrosidase; Parkinson’s disease; Gaucher’s disease; pharmacological chaperones
7.  Quantification of the Influence of Protein-Protein Interactions on Adsorbed Protein Structure and Bioactivity 
While protein-surface interactions have been widely studied, relatively little is understood at this time regarding how protein-surface interaction effects are influenced by protein-protein interactions and how these effects combine with the internal stability of a protein to influence its adsorbed-state structure and bioactivity. The objectives of this study were to develop a method to study these combined effects under widely varying protein-protein interaction conditions using hen egg-white lysozyme (HEWL) adsorbed on silica glass, poly(methyl methacrylate), and polyethylene as our model systems. In order to vary protein-protein interaction effects over a wide range, HEWL was first adsorbed to each surface type under widely varying protein solution concentrations for 2 h to saturate the surface, followed by immersion in pure buffer solution for 15 h to equilibrate the adsorbed protein layers in the absence of additionally adsorbing protein. Periodic measurements were made at selected time points of the areal density of the adsorbed protein layer as an indicator of the level of protein-protein interaction effects within the layer, and these values were then correlated with measurements of the adsorbed protein’s secondary structure and bioactivity. The results from these studies indicate that protein-protein interaction effects help stabilize the structure of HEWL adsorbed on silica glass, have little influence on the structural behavior of HEWL on HDPE, and actually serve to destabilize HEWL’s structure on PMMA. The bioactivity of HEWL on silica glass and HDPE was found to decrease in direct proportion to the degree of adsorption-induce protein unfolding. A direct correlation between bioactivity and the conformational state of adsorbed HEWL was less apparent on PMMA, thus suggesting that other factors influenced HEWL’s bioactivity on this surface, such as the accessibility of HEWL’s bioactive site being blocked by neighboring proteins or the surface itself. The developed methods provide an effective means to characterize the influence of protein-protein interaction effects and provide new molecular-level insights into how protein-protein interaction effects combine with protein-surface interaction and internal protein stability effects to influence the structure and bioactivity of adsorbed protein.
doi:10.1016/j.colsurfb.2013.04.018
PMCID: PMC3732647  PMID: 23751416
8.  Comprehensive Experimental and Computational Analysis of Binding Energy Hot Spots at the NF-κB Essential Modulator (NEMO)/IKKβ Protein-Protein Interface 
We report a comprehensive analysis of binding energy hot spots at the protein-protein interaction (PPI) interface between NF-κB Essential Modulator (NEMO) and IκB kinase subunit β (IKKβ), an interaction that is critical for NF-κB pathway signaling, using experimental alanine scanning mutagenesis and also the FTMap method for computational fragment screening. The experimental results confirm that the previously identified NBD region of IKKβ contains the highest concentration of hot spot residues, the strongest of which are W739, W741 and L742 (ΔΔG = 4.3, 3.5 and 3.2 kcal/mol, respectively). The region occupied by these residues defines a potentially druggable binding site on NEMO that extends for ~16 Å to additionally include the regions that bind IKKβ L737 and F734. NBD residues D738 and S740 are also important for binding but do not make direct contact with NEMO, instead likely acting to stabilize the active conformation of surrounding residues. We additionally found two previously unknown hot spot regions centered on IKKβ residues L708/V709 and L719/I723. The computational approach successfully identified all three hot spot regions on IKKβ. Moreover, the method was able to accurately quantify the energetic importance of all hot spots residues involving direct contact with NEMO. Our results provide new information to guide the discovery of small molecule inhibitors that target the NEMO/IKKβ interaction. They additionally clarify the structural and energetic complementarity between “pocket-forming” and “pocket occupying” hot spot residues, and further validate computational fragment mapping as a method for identifying hot spots at PPI interfaces.
doi:10.1021/ja400914z
PMCID: PMC3680600  PMID: 23506214
IKKγ; alanine scanning mutagenesis; protein-protein interactions; IKKγ; fluorescence polarization; fluorescence anisotropy
9.  Room-temperature X-ray diffraction studies of cisplatin and carboplatin binding to His15 of HEWL after prolonged chemical exposure 
Acta Crystallographica Section F  2012;68(Pt 11):1300-1306.
Binding of cisplatin to His15 in hen egg-white lysozyme in aqueous media is observed after prolonged chemical exposure for 15 months, in contrast to the lack of binding that was observed after 4 d in a previous study. Binding of carboplatin is seen in greater detail in the case of room-temperature data collection compared with cryo data collection.
The anticancer complexes cisplatin and carboplatin are known to bind to both the Nδ and the N∊ atoms of His15 of hen egg-white lysozyme (HEWL) in the presence of dimethyl sulfoxide (DMSO). However, neither binds in aqueous media after 4 d of crystallization and crystal growth, suggesting that DMSO facilitates cisplatin/carboplatin binding to the N atoms of His15 by an unknown mechanism. Crystals of HEWL cocrystallized with cisplatin in both aqueous and DMSO media, of HEWL cocrystallized with carboplatin in DMSO medium and of HEWL cocrystallized with cisplatin and N-acetylglucosamine (NAG) in DMSO medium were stored for between seven and 15 months. X-ray diffraction studies of these crystals were carried out on a Bruker APEX II home-source diffractometer at room temperature. Room-temperature X-ray diffraction data collection removed the need for cryoprotectants to be used, ruling out any effect that the cryoprotectants might have had on binding to the protein. Both cisplatin and carboplatin still bind to both the Nδ and N∊ atoms of His15 in DMSO media as expected, but more detail for the cyclobutanedicarboxylate (CBDC) moiety of carboplatin was observed at the N∊ binding site. However, two molecules of cisplatin were now observed to be bound to His15 in aqueous conditions. The platinum peak positions were identified using anomalous difference electron-density maps as a cross-check with F o − F c OMIT electron-density maps. The occupancies of each binding site were calculated using SHELXTL. These results show that over time cisplatin binds to both N atoms of His15 of HEWL in aqueous media, whereas this binding is speeded up in the presence of DMSO. The implication of cisplatin binding to proteins after a prolonged period of time is an important consideration for the length of treatment in patients who are given cisplatin.
doi:10.1107/S1744309112042005
PMCID: PMC3515368  PMID: 23143236
cisplatin; carboplatin; histidine; aqueous media; DMSO media; data collection at room temperature; data collection with ω versus ϕ scans with capillaries
10.  The active site of hen egg-white lysozyme: flexibility and chemical bonding 
Chemical bonding at the active site of lysozyme is analyzed on the basis of a multipole model employing transferable multipole parameters from a database. Large B factors at low temperatures reflect frozen-in disorder, but therefore prevent a meaningful free refinement of multipole parameters.
Chemical bonding at the active site of hen egg-white lysozyme (HEWL) is analyzed on the basis of Bader’s quantum theory of atoms in molecules [QTAIM; Bader (1994 ▶), Atoms in Molecules: A Quantum Theory. Oxford University Press] applied to electron-density maps derived from a multipole model. The observation is made that the atomic displacement parameters (ADPs) of HEWL at a temperature of 100 K are larger than ADPs in crystals of small biological molecules at 298 K. This feature shows that the ADPs in the cold crystals of HEWL reflect frozen-in disorder rather than thermal vibrations of the atoms. Directly generalizing the results of multipole studies on small-molecule crystals, the important consequence for electron-density analysis of protein crystals is that multipole parameters cannot be independently varied in a meaningful way in structure refinements. Instead, a multipole model for HEWL has been developed by refinement of atomic coordinates and ADPs against the X-ray diffraction data of Wang and coworkers [Wang et al. (2007), Acta Cryst. D63, 1254–1268], while multipole parameters were fixed to the values for transferable multipole parameters from the ELMAM2 database [Domagala et al. (2012), Acta Cryst. A68, 337–351] . Static and dynamic electron densities based on this multipole model are presented. Analysis of their topological properties according to the QTAIM shows that the covalent bonds possess similar properties to the covalent bonds of small molecules. Hydrogen bonds of intermediate strength are identified for the Glu35 and Asp52 residues, which are considered to be essential parts of the active site of HEWL. Furthermore, a series of weak C—H⋯O hydrogen bonds are identified by means of the existence of bond critical points (BCPs) in the multipole electron density. It is proposed that these weak interactions might be important for defining the tertiary structure and activity of HEWL. The deprotonated state of Glu35 prevents a distinction between the Phillips and Koshland mechanisms.
doi:10.1107/S1399004714001928
PMCID: PMC3975892  PMID: 24699657
hen egg-white lysozyme; multipole model; multipole parameters
11.  Carboplatin binding to histidine 
An X-ray crystal structure showing the binding of purely carboplatin to histidine in a model protein has finally been obtained. This required extensive crystallization trials and various novel crystal structure analyses.
Carboplatin is a second-generation platinum anticancer agent used for the treatment of a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine (in hen egg-white lysozyme; HEWL) showed the partial conversion of carboplatin to cisplatin owing to the high NaCl concentration used in the crystallization conditions. HEWL co-crystallizations with carboplatin in NaBr conditions have now been carried out to confirm whether carboplatin converts to the bromine form and whether this takes place in a similar way to the partial conversion of carboplatin to cisplatin observed previously in NaCl conditions. Here, it is reported that a partial chemical transformation takes place but to a transplatin form. Thus, to attempt to resolve purely carboplatin binding at histidine, this study utilized co-crystallization of HEWL with carboplatin without NaCl to eliminate the partial chemical conversion of carboplatin. Tetragonal HEWL crystals co-crystallized with carboplatin were successfully obtained in four different conditions, each at a different pH value. The structural results obtained show carboplatin bound to either one or both of the N atoms of His15 of HEWL, and this particular variation was dependent on the concentration of anions in the crystallization mixture and the elapsed time, as well as the pH used. The structural details of the bound carboplatin molecule also differed between them. Overall, the most detailed crystal structure showed the majority of the carboplatin atoms bound to the platinum centre; however, the four-carbon ring structure of the cyclobutanedicarboxylate moiety (CBDC) remained elusive. The potential impact of the results for the administration of carboplatin as an anticancer agent are described.
doi:10.1107/S2053230X14016161
PMCID: PMC4157408  PMID: 25195881
carboplatin; histidine; avoid partial conversion to cisplatin; NaBr crystallization conditions; non-NaCl crystallization conditions; model protein (hen egg-white lysozyme)
12.  Improving Protocols for Protein Mapping through Proper Comparison to Crystallography Data 
Computational approaches to fragment-based drug design (FBDD) can complement experiments and facilitate the identification of potential hot spots along the protein surface. However, the evaluation of computational methods for mapping binding sites frequently focuses upon the ability to reproduce crystallographic coordinates to within a low RMSD threshold. This dependency on the deposited coordinate data overlooks the original electron density from the experiment, thus techniques may be developed based upon subjective - or even erroneous - atomic coordinates. This can become a significant drawback in applications to systems where the location of hot spots is unknown. Based on comparison to crystallographic density, we previously showed that mixed-solvent molecular dynamics (MixMD) accurately identifies the active site for HEWL, with acetonitrile as an organic solvent. Here, we concentrated on the influence of protic solvent on simulation and refined the optimal MixMD approach for extrapolation of the method to systems without established sites. Our results establish an accurate approach for comparing simulations to experiment. We have outlined the most efficient strategy for MixMD, based on simulation length and number of runs. The development outlined here makes MixMD a robust method which should prove useful across a broad range of target structures. Lastly, our results with MixMD match experimental data so well that consistency between simulations and density may be a useful way to aid the identification of probes vs waters during the refinement of future MSCS crystallographic structures.
doi:10.1021/ci300430v
PMCID: PMC3581705  PMID: 23327200
Molecular dynamics; hot spots; solvent competition; protein flexibility; fragment-based drug discovery
13.  Mapping the Druggable Allosteric Space of G-Protein Coupled Receptors: a Fragment-Based Molecular Dynamics Approach 
Chemical biology & drug design  2010;76(3):201-217.
To address the problem of specificity in G-protein coupled receptor (GPCR) drug discovery, there has been tremendous recent interest in allosteric drugs that bind at sites topographically distinct from the orthosteric site. Unfortunately, structure-based drug design of allosteric GPCR ligands has been frustrated by the paucity of structural data for allosteric binding sites, making a strong case for predictive computational methods. In this work, we map the surfaces of the β1 (β1AR) and β2 (β2AR) adrenergic receptor structures, to detect a series of five potentially druggable allosteric sites. We employ the FTMAP algorithm to identify “hot spots” with affinity for a variety of organic probe molecules corresponding to drug fragments. Our work is distinguished by an ensemble-based approach, whereby we map diverse receptor conformations taken from Molecular Dynamics (MD) simulations totalling ~0.5 μs. Our results reveal distinct pockets formed at both solvent-exposed and lipid-exposed cavities, which we interpret in the light of experimental data and which may constitute novel targets for GPCR drug discovery. This mapping data can now serve to drive a combination of fragment-based and virtual screening approaches for the discovery of small molecules that bind at these sites and which may offer highly selective therapies.
doi:10.1111/j.1747-0285.2010.01012.x
PMCID: PMC2918726  PMID: 20626410
molecular dynamics; allosteric; GPCR; docking; fragment-based
14.  Mapping the Druggable Allosteric Space of G-Protein Coupled Receptors: a Fragment-Based Molecular Dynamics Approach 
Chemical Biology & Drug Design  2010;76(3):201-217.
To address the problem of specificity in G-protein coupled receptor (GPCR) drug discovery, there has been tremendous recent interest in allosteric drugs that bind at sites topographically distinct from the orthosteric site. Unfortunately, structure-based drug design of allosteric GPCR ligands has been frustrated by the paucity of structural data for allosteric binding sites, making a strong case for predictive computational methods. In this work, we map the surfaces of the β1 (β1AR) and β2 (β2AR) adrenergic receptor structures to detect a series of five potentially druggable allosteric sites. We employ the FTMAP algorithm to identify ‘hot spots’ with affinity for a variety of organic probe molecules corresponding to drug fragments. Our work is distinguished by an ensemble-based approach, whereby we map diverse receptor conformations taken from molecular dynamics (MD) simulations totaling approximately 0.5 μs. Our results reveal distinct pockets formed at both solvent-exposed and lipid-exposed cavities, which we interpret in light of experimental data and which may constitute novel targets for GPCR drug discovery. This mapping data can now serve to drive a combination of fragment-based and virtual screening approaches for the discovery of small molecules that bind at these sites and which may offer highly selective therapies.
doi:10.1111/j.1747-0285.2010.01012.x
PMCID: PMC2918726  PMID: 20626410
allosteric; docking; fragment-based; GPCR; molecular dynamics
15.  FTFlex: accounting for binding site flexibility to improve fragment-based identification of druggable hot spots 
Bioinformatics  2013;29(9):1218-1219.
Computational solvent mapping finds binding hot spots, determines their druggability and provides information for drug design. While mapping of a ligand-bound structure yields more accurate results, usually the apo structure serves as the starting point in design. The FTFlex algorithm, implemented as a server, can modify an apo structure to yield mapping results that are similar to those of the respective bound structure. Thus, FTFlex is an extension of our FTMap server, which only considers rigid structures. FTFlex identifies flexible residues within the binding site and determines alternative conformations using a rotamer library. In cases where the mapping results of the apo structure were in poor agreement with those of the bound structure, FTFlex was able to yield a modified apo structure, which lead to improved FTMap results. In cases where the mapping results of the apo and bound structures were in good agreement, no new structure was predicted.
Availability: FTFlex is freely available as a web-based server at http://ftflex.bu.edu/.
Contact: vajda@bu.edu or midas@bu.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btt102
PMCID: PMC3634182  PMID: 23476022
16.  The Two Sides of Complement C3d: Evolution of Electrostatics in a Link between Innate and Adaptive Immunity 
PLoS Computational Biology  2012;8(12):e1002840.
The interaction between complement fragment C3d and complement receptor 2 (CR2) is a key aspect of complement immune system activation, and is a component in a link between innate and adaptive immunities. The complement immune system is an ancient mechanism for defense, and can be found in species that have been on Earth for the last 600 million years. However, the link between the complement system and adaptive immunity, which is formed through the association of the B-cell co-receptor complex, including the C3d-CR2 interaction, is a much more recent adaptation. Human C3d and CR2 have net charges of −1 and +7 respectively, and are believed to have evolved favoring the role of electrostatics in their functions. To investigate the role of electrostatics in the function and evolution of human C3d and CR2, we have applied electrostatic similarity methods to identify regions of evolutionarily conserved electrostatic potential based on 24 homologues of complement C3d and 4 homologues of CR2. We also examine the effects of structural perturbation, as introduced through molecular dynamics and mutations, on spatial distributions of electrostatic potential to identify perturbation resistant regions, generated by so-called electrostatic “hot-spots”. Distributions of electrostatic similarity based on families of perturbed structures illustrate the presence of electrostatic “hot-spots” at the two functional sites of C3d, while the surface of CR2 lacks electrostatic “hot-spots” despite its excessively positive nature. We propose that the electrostatic “hot-spots” of C3d have evolved to optimize its dual-functionality (covalently attaching to pathogen surfaces and interaction with CR2), which are both necessary for the formation B-cell co-receptor complexes. Comparison of the perturbation resistance of the electrostatic character of the homologues of C3d suggests that there was an emergence of a new role of electrostatics, and a transition in the function of C3d, after the divergence of jawless fish.
Author Summary
Complement fragment C3d is a thioester-containing protein that is a key component/domain in the complement system, an ancient line of defense, due to its ability to covalently attach to pathogen cell surfaces, such as bacteria. As the immune system evolved in complexity, from acellular defense mechanisms to multicellular systems with memory, so has the function of C3d. In humans, but not lower species such as invertebrates, C3d attached to pathogen surfaces binds B-cell co-receptor CR2, in conjunction with an antibody/antigen complex, forming a link between the innate and adaptive immune systems. The C3d-CR2 interaction ultimately increases B-cell sensitivity to the C3d tagged pathogen by 1,000–10,000 fold, and is known to be driven by electrostatic forces. Since electrostatics are crucial to the C3d-CR2 interaction, it is likely that probing the evolution of the electrostatics of C3d and CR2 will provide insight into this gained function. To this end, we employ a novel computational approach for identifying the electrostatic “hot-spots” of C3d and CR2, which are produced by clusters of like-charged residues found on the surface of the protein. Electrostatic “hot-spots” are often evolutionarily favored and in this study provide new insight into the evolution of C3d in its role in a link between innate and adaptive immunity.
doi:10.1371/journal.pcbi.1002840
PMCID: PMC3531323  PMID: 23300422
17.  Expanding the Druggable Space of the LSD1/CoREST Epigenetic Target: New Potential Binding Regions for Drug-Like Molecules, Peptides, Protein Partners, and Chromatin 
PLoS Computational Biology  2013;9(7):e1003158.
Lysine specific demethylase-1 (LSD1/KDM1A) in complex with its corepressor protein CoREST is a promising target for epigenetic drugs. No therapeutic that targets LSD1/CoREST, however, has been reported to date. Recently, extended molecular dynamics (MD) simulations indicated that LSD1/CoREST nanoscale clamp dynamics is regulated by substrate binding and highlighted key hinge points of this large-scale motion as well as the relevance of local residue dynamics. Prompted by the urgent need for new molecular probes and inhibitors to understand LSD1/CoREST interactions with small-molecules, peptides, protein partners, and chromatin, we undertake here a configurational ensemble approach to expand LSD1/CoREST druggability. The independent algorithms FTMap and SiteMap and our newly developed Druggable Site Visualizer (DSV) software tool were used to predict and inspect favorable binding sites. We find that the hinge points revealed by MD simulations at the SANT2/Tower interface, at the SWIRM/AOD interface, and at the AOD/Tower interface are new targets for the discovery of molecular probes to block association of LSD1/CoREST with chromatin or protein partners. A fourth region was also predicted from simulated configurational ensembles and was experimentally validated to have strong binding propensity. The observation that this prediction would be prevented when using only the X-ray structures available (including the X-ray structure bound to the same peptide) underscores the relevance of protein dynamics in protein interactions. A fifth region was highlighted corresponding to a small pocket on the AOD domain. This study sets the basis for future virtual screening campaigns targeting the five novel regions reported herein and for the design of LSD1/CoREST mutants to probe LSD1/CoREST binding with chromatin and various protein partners.
Author Summary
Protein dynamics plays a major role in determining the molecular interactions available to molecular binding partners, including druggable hot spots. The LSD1/CoREST complex is one of the most relevant epigenetic targets discovered and was shown to be a highly dynamic nanoscale clamp using molecular dynamics simulations. The general relationship between LSD1/CoREST dynamics and the molecular sites available for non-covalent interactions with an array of known binding partners (from relatively small drug-like molecules and peptides, to larger proteins and chromatin) remains relatively unexplored. We employed an integrated experimental and computational biology approach to effectively capture the nature of non-covalent binding interactions available to the LSD1/CoREST nanoscale complex. This ensemble approach relies on the newly developed graphical visualization by Druggable Site Visualizer (DSV) that allows treatment of large-size protein configurational ensembles data and is freely distributed to the public and readily transferable to other protein targets of pharmacological interest.
doi:10.1371/journal.pcbi.1003158
PMCID: PMC3715402  PMID: 23874194
18.  Binding Structures of tri-N-acetyl-β-glucosamine in Hen Egg White Lysozyme using Molecular Dynamics with a Polarizable Force Field 
Lysozyme is a well-studied enzyme that hydrolyzes the β-(1,4)-glycosidic linkage of N-acetyl-β-glucosamine (NAG)n oligomers. The active site of hen egg-white lysozyme (HEWL) is believed to consist of six subsites, A-F that can accommodate six sugar residues. We present studies exploring the use of polarizable force fields in conjunction with all-atom molecular dynamics simulations to analyze binding structures of complexes of lysozyme and NAG trisaccharide, (NAG)3. Molecular dynamics trajectories are applied to analyze structures and conformation of the complex as well as protein-ligand interactions, including the hydrogen-bonding network in the binding pocket. Two binding modes (ABC and BCD) of (NAG)3 are investigated independently based on a fixed-charge model and a polarizable model. We also apply MM-GBSA methods based on molecular dynamics using both non-polarizable and polarizable force fields in order to compute binding free energies. We also study the correlation between RMSD and binding free energies of the wildtype and W62Y mutant; we find that for this prototypical system, approaches using the MD trajectories coupled with implicit solvent models are equivalent for polarizable and fixed-charge models.
doi:10.1002/jcc.23109
PMCID: PMC4214157  PMID: 23109228
19.  Chemical conversion of cisplatin and carboplatin with histidine in a model protein crystallized under sodium iodide conditions 
Crystals of HEWL with cisplatin and HEWL with carboplatin grown in sodium iodide conditions both show a partial chemical transformation of cisplatin or carboplatin to a transiodoplatin (PtI2 X 2) form. The binding is only at the Nδ atom of His15. A further Pt species (PtI3 X) is also seen, in both cases bound in a crevice between symmetry-related protein molecules.
Cisplatin and carboplatin are platinum anticancer agents that are used to treat a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine in hen egg-white lysozyme (HEWL) showed a partial chemical conversion of carboplatin to cisplatin owing to the high sodium chloride concentration used in the crystallization conditions. Also, the co-crystallization of HEWL with carboplatin in sodium bromide conditions resulted in the partial conversion of carboplatin to the transbromoplatin form, with a portion of the cyclobutanedicarboxylate (CBDC) moiety still present. The results of the co-crystallization of HEWL with cisplatin or carboplatin in sodium iodide conditions are now reported in order to determine whether the cisplatin and carboplatin converted to the iodo form, and whether this took place in a similar way to the partial conversion of carboplatin to cisplatin in NaCl conditions or to transbromoplatin in NaBr conditions as seen previously. It is reported here that a partial chemical transformation has taken place to a transplatin form for both ligands. The NaI-grown crystals belonged to the monoclinic space group P21 with two molecules in the asymmetric unit. The chemically transformed cisplatin and carboplatin bind to both His15 residues, i.e. in each asymmetric unit. The binding is only at the Nδ atom of His15. A third platinum species is also seen in both conditions bound in a crevice between symmetry-related molecules. Here, the platinum is bound to three I atoms identified based on their anomalous difference electron densities and their refined occupancies, with the fourth bound atom being a Cl atom (in the cisplatin case) or a portion of the CBDC moiety (in the carboplatin case).
doi:10.1107/S2053230X14013995
PMCID: PMC4157406  PMID: 25195879
cisplatin; carboplatin; sodium iodide crystallization; histidine; transiodoplatin (PtI2X2); PtI3X species
20.  APIS: accurate prediction of hot spots in protein interfaces by combining protrusion index with solvent accessibility 
BMC Bioinformatics  2010;11:174.
Background
It is well known that most of the binding free energy of protein interaction is contributed by a few key hot spot residues. These residues are crucial for understanding the function of proteins and studying their interactions. Experimental hot spots detection methods such as alanine scanning mutagenesis are not applicable on a large scale since they are time consuming and expensive. Therefore, reliable and efficient computational methods for identifying hot spots are greatly desired and urgently required.
Results
In this work, we introduce an efficient approach that uses support vector machine (SVM) to predict hot spot residues in protein interfaces. We systematically investigate a wide variety of 62 features from a combination of protein sequence and structure information. Then, to remove redundant and irrelevant features and improve the prediction performance, feature selection is employed using the F-score method. Based on the selected features, nine individual-feature based predictors are developed to identify hot spots using SVMs. Furthermore, a new ensemble classifier, namely APIS (A combined model based on Protrusion Index and Solvent accessibility), is developed to further improve the prediction accuracy. The results on two benchmark datasets, ASEdb and BID, show that this proposed method yields significantly better prediction accuracy than those previously published in the literature. In addition, we also demonstrate the predictive power of our proposed method by modelling two protein complexes: the calmodulin/myosin light chain kinase complex and the heat shock locus gene products U and V complex, which indicate that our method can identify more hot spots in these two complexes compared with other state-of-the-art methods.
Conclusion
We have developed an accurate prediction model for hot spot residues, given the structure of a protein complex. A major contribution of this study is to propose several new features based on the protrusion index of amino acid residues, which has been shown to significantly improve the prediction performance of hot spots. Moreover, we identify a compact and useful feature subset that has an important implication for identifying hot spot residues. Our results indicate that these features are more effective than the conventional evolutionary conservation, pairwise residue potentials and other traditional features considered previously, and that the combination of our and traditional features may support the creation of a discriminative feature set for efficient prediction of hot spot residues. The data and source code are available on web site http://home.ustc.edu.cn/~jfxia/hotspot.html.
doi:10.1186/1471-2105-11-174
PMCID: PMC2874803  PMID: 20377884
21.  Residual dipolar couplings: are multiple independent alignments always possible? 
Journal of Biomolecular Nmr  2010;49(1):53-60.
RDCs for the 14 kDa protein hen egg-white lysozyme (HEWL) have been measured in eight different alignment media. The elongated shape and strongly positively charged surface of HEWL appear to limit the protein to four main alignment orientations. Furthermore, low levels of alignment and the protein’s interaction with some alignment media increases the experimental error. Together with heterogeneity across the alignment media arising from constraints on temperature, pH and ionic strength for some alignment media, these data are suitable for structure refinement, but not the extraction of dynamic parameters. For an analysis of protein dynamics the data must be obtained with very low errors in at least three or five independent alignment media (depending on the method used) and so far, such data have only been reported for three small 6–8 kDa proteins with identical folds: ubiquitin, GB1 and GB3. Our results suggest that HEWL is likely to be representative of many other medium to large sized proteins commonly studied by solution NMR. Comparisons with over 60 high-resolution crystal structures of HEWL reveal that the highest resolution structures are not necessarily always the best models for the protein structure in solution.
doi:10.1007/s10858-010-9457-1
PMCID: PMC3020303  PMID: 21184138
Residual dipolar couplings; Hen egg-white lysozyme; Dynamics; Protein alignment; Ubiquitin
22.  FAMBE-pH: A Fast and Accurate Method to Compute the Total Solvation Free Energies of Proteins 
The journal of physical chemistry. B  2008;112(35):11122-11136.
A fast and accurate method to compute the total solvation free energies of proteins as a function of pH is presented. The method makes use of a combination of approaches, some of which have already appeared in the literature; (i) the Poisson equation is solved with an optimized fast adaptive multigrid boundary element (FAMBE) method; (ii) the electrostatic free energies of the ionizable sites are calculated for their neutral and charged states by using a detailed model of atomic charges; (iii) a set of optimal atomic radii is used to define a precise dielectric surface interface; (iv) a multilevel adaptive tessellation of this dielectric surface interface is achieved by using multisized boundary elements; and (v) 1:1 salt effects are included. The equilibrium proton binding/release is calculated with the Tanford–Schellman integral if the proteins contain more than ∼20–25 ionizable groups; for a smaller number of ionizable groups, the ionization partition function is calculated directly. The FAMBE method is tested as a function of pH (FAMBE-pH) with three proteins, namely, bovine pancreatic trypsin inhibitor (BPTI), hen egg white lysozyme (HEWL), and bovine pancreatic ribonuclease A (RNaseA). The results are (a) the FAMBE-pH method reproduces the observed pKa's of the ionizable groups of these proteins within an average absolute value of 0.4 pK units and a maximum error of 1.2 pK units and (b) comparison of the calculated total pH-dependent solvation free energy for BPTI, between the exact calculation of the ionization partition function and the Tanford–Schellman integral method, shows agreement within 1.2 kcal/mol. These results indicate that calculation of total solvation free energies with the FAMBE-pH method can provide an accurate prediction of protein conformational stability at a given fixed pH and, if coupled with molecular mechanics or molecular dynamics methods, can also be used for more realistic studies of protein folding, unfolding, and dynamics, as a function of pH.
doi:10.1021/jp709969n
PMCID: PMC2760452  PMID: 18683966
23.  Crowding induced collective hydration of biological macromolecules over extended distances 
Ultrafast two-dimensional infrared (2D-IR) spectroscopy reveals picosecond protein and hydration dynamics of crowded hen egg white lysozyme (HEWL) labeled with a metal-carbonyl vibrational probe covalently attached to a solvent accessible His residue. HEWL is systematically crowded alternatively with polyethylene glycol (PEG) or excess lysozyme in order to distinguish the chemically inert polymer from the complex electrostatic profile of the protein crowder. The results are threefold: (1) A sharp dynamical jamming-like transition is observed in the picosecond protein and hydration dynamics that is attributed to an independent-to-collective hydration transition induced by macromolecular crowding that slows the hydration dynamics up to an order of magnitude relative to bulk water; (2) The interprotein distance at which the transition occurs suggests collective hydration of proteins over distances of 30-40 Å; and (3) Comparing the crowding effects of PEG400 to our previously reported experiments using glycerol exposes fundamental differences between small and macromolecular crowding agents.
doi:10.1021/ja407858c
PMCID: PMC3983708  PMID: 24341684
24.  The structural basis of pregnane X receptor binding promiscuity 
Biochemistry  2009;48(48):11572-11581.
The steroid and xenobiotic-responsive human pregnane X receptor (PXR) binds a broad range of structurally diverse compounds. The structures of the apo and ligand-bound forms of PXR are very similar, in contrast to most promiscuous proteins that generally adapt their shape to different ligands. We investigated the structural origins of PXR's recognition promiscuity using computational solvent mapping, a technique developed for the identification and characterization of hot spots, i.e., regions of the protein surface that are major contributors to the binding free energy. Results reveal that the smooth and nearly spherical binding site of PXR has a well-defined hot spot structure, with four hot spots located on four different sides of the pocket and a fifth close to its center. Three of these hot spots are already present in the ligand-free protein. The most important hot spot is defined by three structurally and sequentially conserved residues, W299, F288, and Y306. This largely hydrophobic site is not very specific, and interacts with all known PXR ligands. Depending on their sizes and shapes, individual PXR ligands extend into 2, 3, or 4 more hot spot regions. The large number of potential arrangements within the binding site explains why PXR is able to accommodate a large variety of compounds. All five hot spots include at least one important residue, which is conserved in all mammalian PXRs, suggesting that the hot spot locations have remained largely invariant during mammalian evolution. The same side chains also show a high level of structural conservation across hPXR structures. However, each of the hPXR hot spots also includes residues with moveable side chains, further increasing the size variation in ligands that PXR can bind. Results also suggest a unique signal transduction mechanism between the PXR homodimerization interface and its co-activator binding site.
doi:10.1021/bi901578n
PMCID: PMC2789303  PMID: 19856963
25.  Differentiation among closely related organisms of the Actinobacillus-Haemophilus-Pasteurella group by means of lysozyme and EDTA. 
Journal of Clinical Microbiology  1985;22(4):629-636.
Bacteriolysis in Tris-maleate buffer (0.005 M, pH 7.2) supplemented with EDTA (0.01 M) and hen egg white lysozyme (HEWL, 1.0 microgram/ml) was set up to assist differentiation between the taxonomically closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. A. actinomycetemcomitans was more sensitive to lysis in this system than H. aphrophilus. The standard method for bacteriolysis separated the 10 tested strains of A. actinomycetemcomitans into two groups (I and II) based on their lysis patterns, whereas the 7 strains of H. aphrophilus examined were homogeneous. In group I of A. actinomycetemcomitans, EDTA displayed a considerable lytic effect, which was not increased by supplementation with HEWL. In group II, the lytic effect of EDTA was much less, but HEWL had a considerable supplementary lytic effect. When the turbidity of A. actinomycetemcomitans (ATCC 29522) or H. aphrophilus (ATCC 33389) suspended in Tris buffer was monitored at close pH intervals (0.2) from pH 5.2 to 9.2, maximal lysis of ATCC 29522 occurred with EDTA at pH 8.0 and with EDTA-HEWL at pH 7.6, while ATCC 33389 lysed with EDTA at pH 9.0 and with EDTA-HEWL at pH 9.2. When other members of the family Pasteurellaceae (Haemophilus influenzae type b, Haemophilus paraphrophilus, Pasteurella multocida, Pasteurella haemolytica, and Pasteurella ureae) were included for comparison, the group I strains of A. actinomycetemcomitans were the most rapidly lysed by EDTA. H. paraphrophilus was the least sensitive of the gram-negative strains tested, but not as resistant as Micrococcus luteus (control). M. luteus was the organism most sensitive to lysozyme, followed by P. ureae and the group II strains of A. actinomycetemcomitans, while the group I strains of A. actinomycetemcomitans, H. paraphrophilus, and P. haemolytica were the least sensitive organisms.
Images
PMCID: PMC268481  PMID: 3935663

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