Functional definition of the tobacco protoporphyrinogen IX oxidase substrate-binding site

PPO (protoporphyrinogen IX oxidase) catalyses the flavin-dependent six-electron oxidation of protogen (protoporphyrinogen IX) to form proto (protoporphyrin IX), a crucial step in haem and chlorophyll biosynthesis. The apparent K(m) value for wild-type tobacco PPO2 (mitochondrial PPO) was 1.17 muM, with a V(max) of 4.27 muM.min(-1).mg(-1) and a catalytic activity k(cat) of 6.0 s(-1). Amino acid residues that appear important for substrate binding in a crystal structure-based model of the substrate docked in the active site were interrogated by site-directed mutagenesis. PPO2 variant F392H did not reveal detectable enzyme activity indicating an important role of Phe(392) in substrate ring A stacking. Mutations of Leu(356), Leu(372) and Arg(98) increased k(cat) values up to 100-fold, indicating that the native residues are not essential for establishing an orientation of the substrate conductive to catalysis. Increased K(m) values of these PPO2 variants from 2- to 100-fold suggest that these residues are involved in, but not essential to, substrate binding via rings B and C. Moreover, one prominent structural constellation of human PPO causing the disease variegate porphyria (N67W/S374D) was successfully transferred into the tobacco PPO2 background. Therefore tobacco PPO2 represents a useful model system for the understanding of the structure-function relationship underlying detrimental human enzyme defects.     

Computational studies of the farnesyltransferase ternary complex part I: substrate binding

Farnesyltransferase (FTase) catalyzes the transfer of farnesyl from farnesyl diphosphate (FPP) to cysteine residues at or near the C-terminus of protein acceptors with a CaaX motif (a, aliphatic; X, Met). Farnesylation is a critical modification to many switch proteins involved in the extracellular signal transduction pathway, which facilitates their fixation on the cell membrane where the extracellular signal is processed. The malfunction caused by mutations in these proteins often causes uncontrolled cell reproduction and leads to tumor formation. FTase inhibitors have been extensively studied as potential anticancer agents in recent years, several of which have advanced to different phases of clinical trials. However, the mechanism of this biologically important enzyme has not been firmly established. Understanding how FTase recruits the FPP substrate is the first and foremost step toward further mechanistic investigations and the design of effective FTase inhibitors. Molecular dynamic simulations were carried out on the ternary structure of FTase complexed with the FPP substrate and an acetyl-capped tetrapeptide (acetyl-CVIM), which revealed that the FPP substrate maintains an inactive conformation and the binding of the diphosphate group can be largely attributed to residues R291beta, K164alpha, K294beta, and H248beta. The FPP substrate assumes an extended conformation in the binding site with restricted rotation of the backbone dihedral angles; however, it does not have a well-defined conformation when unbound in solution. This is evident from multinanosecond MD simulations of the FPP substrate in a vacuum and solution. Our conclusion is further supported by theoretical J coupling calculations. Our results on the FPP binding are in good agreement with previous experimental kinetic studies on FTase mutants. The hypothetical conformational activation of the FPP substrate is currently under investigation.     

Protonation of Glu135 Facilitates the Outward-to-Inward Structural Transition of Fucose Transporter

Major facilitator superfamily (MFS) transporters typically need to alternatingly sample the outward-facing and inward-facing conformations, in order to transport the substrate across membrane. To understand the mechanism, in this work, we focused on one MFS member, the L-fucose/H⁺ symporter (FucP), whose crystal structure exhibits an outward-open conformation. Previous experiments imply several residues critical to the substrate/proton binding and structural transition of FucP, among which Glu¹³⁵, located in the periplasm-accessible vestibule, is supposed as being involved in both proton translocation and conformational change of the protein. Here, the structural transition of FucP in presence of substrate was investigated using molecular-dynamics simulations. By combining the equilibrium and accelerated simulations as well as thermodynamic calculations, not only was the large-scale conformational change from the outward-facing to inward-facing state directly observed, but also the free energy change during the structural transition was calculated. The simulations confirm the critical role of Glu¹³⁵, whose protonation facilitates the outward-to-inward structural transition both by energetically favoring the inward-facing conformation in thermodynamics and by reducing the free energy barrier along the reaction pathway in kinetics. Our results may help the mechanistic studies of both FucP and other MFS transporters.     

Site-directed mutagenesis and computational study of the Y366 active site in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> protoporphyrinogen oxidase

Protoporphyrinogen IX oxidase (PPO), the last common enzyme of heme and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX, with FAD as cofactor. Among PPO, Bacillus subtilis PPO (bsPPO) is unique because of its broad substrate specificity and resistance to inhibition by diphenylethers. Identification of the activity of bsPPO would help us to understand the catalysis and resistance mechanisms. Based on the modeling and docking studies, we found that Y366 site in bsPPO was adjacent to substrate and FAD. In order to evaluate the functional role of this site, three mutants Y366A Y366E and Y366H were cloned and kinetically characterized. The efficiency of catalysis for Y366A and Y366H reduced to 10% of the wild-type enzyme’s activity, while Y366E just retained 1%. Y366E shows large resistance (K _i = 153.94 μM) to acifluorfen. Molecular docking was carried out to understand the structure and functional relationship of PPO. The experimental results from the site-directed mutagenesis are consistent with the computational studies. The residue at position 366 is seemed to be responsible for substrate binding and catalysis and involved in herbicide resistance of bsPPO.     

Understanding the relative affinity and specificity of the substrate binding site of protein kinase B for substrate-mimetic inhibitors

Designing selective inhibitor of protein kinase B (PKB/Akt) is an area of intense research to develop potential anticancer drugs. As a general point of strategy, the peptide substrate-binding site only responds to a highly specific sequence of amino acids. Targeting the substrate-mimetic inhibitors to the peptide substrate-binding site has the potential for better selectivity. It is therefore of great interest to understand the peptide substrate binding mode of PKB, as well as its specificity and affinity for different substrate-mimetic inhibitors. In the present study, we used molecular dynamic simulations to better understand the interactions of the PKB substrate-binding site with the substrate-mimetic inhibitors. Our computational models successfully mirrored PKB’s selectivity for the substrate-mimetic inhibitors. Furthermore, the key residues interacting with the substrate-mimetic inhibitor were discussed by analysing the different interaction modes of these inhibitors, with different inhibitory potencies, binding to PKB and by comparing the different binding free energy contributions of corresponding residues around the binding pocket. The pharmacophoric requirements were then also summarised for the substrate-mimetic inhibitor binding to PKB. It is expected that this work will provide useful chemical or biochemical informatics for the design of novel and potent substrate-mimetic inhibitors of PKB.     

Insights into the role of d-amino acid oxidase mutations in amyotrophic lateral sclerosis

Missense mutations in the coding region of d -amino acid oxidase (DAO) have been found in patients suffering from amyotrophic lateral sclerosis (ALS). Mutations primarily impair the enzymatic activity of DAO and cause neurodegeneration due to an abnormal accumulation of d -serine in the spinal cord. However, the structural and dynamic changes that lead to impaired enzymatic activity are not fully understood. We present here extensive molecular dynamics simulations of wild-type, and all reported ALS-associated DAO mutants to elucidate the plausible mechanisms of impaired enzymatic activity, a critical function needed for neuroprotection. Simulation results show that DAO mutations disrupt several key interactions with the active site residues and decrease the conformational flexibility of active site loop comprising 216 to 228 residues, necessary for substrate binding and product release. This conformational restriction of the active site loop in the mutants is mainly due to the distortion of critical salt bridge and hydrogen bond interactions compared with wild-type. Furthermore, binding free energy calculations show that DAO mutants have a lower binding affinity toward cofactor flavin adenine dinucleotide and substrate imino-serine than the wild-type. A closer look at the cofactor and substrate interaction profiles further show that DAO mutants have lost several critical interactions with the neighboring residues as compared with wild-type. Taken together, this study provides first-hand explanation of crucial structural features that lead to the loss of enzymatic function in DAO mutants and highlights the need of further genomic scans of patients with ALS to map the association of novel DAO variants in ALS pathophysiology.     

Catalytic mechanism of the inverting N-acetylglucosaminyltransferase I: DFT quantum mechanical model of the reaction pathway and determination of the transition state structure

The complex N-glycan structures on glycoproteins play important roles in cell adhesion and recognition events in metazoan organisms. A critical step in the biosynthetic pathway leading from high mannose to these complex structures includes the transfer of N-acetylglucosamine (GlcNAc) to a mannose residue by the inverting N-acetylglucosaminyltransferase I (GnT-I). The catalytic mechanism of this enzymatic reaction is explored herein using DFT quantum chemical methods. The computational model used to follow the reaction is based on the X-ray crystallographic structure of GnT-I and contains 127 atoms that represent fragments of residues critical for the substrate binding and catalysis. The mechanism of the catalytic reaction was monitored by means of a 2D potential energy map calculated as a function of predefined reaction coordinates at the B3LYP/6-31G** level. This potential energy surface revealed one transition state associated with a reaction pathway following a concerted mechanism. The reaction barrier was estimated, and the structure of the transition state was characterized at the B3LYP/6-311++G**// B3LYP/6-31G** level.     

Structural insights into the catalytic active site and activity of human Nit2/ω-amidase: kinetic assay and molecular dynamics simulation

Human nitrilase-like protein 2 (hNit2) is a putative tumor suppressor, recently identified as ω-amidase. hNit2/ω-amidase plays a crucial metabolic role by catalyzing the hydrolysis of α-ketoglutaramate (the α-keto analog of glutamine) and α-ketosuccinamate (the α-keto analog of asparagine), yielding α-ketoglutarate and oxaloacetate, respectively. Transamination between glutamine and α-keto-γ-methiolbutyrate closes the methionine salvage pathway. Thus, hNit2/ω-amidase links sulfur metabolism to the tricarboxylic acid cycle. To elucidate the catalytic specificity of hNit2/ω-amidase, we performed molecular dynamics simulations on the wild type enzyme and its mutants to investigate enzyme-substrate interactions. Binding free energies were computed to characterize factors contributing to the substrate specificity. The predictions resulting from these computations were verified by kinetic analyses and mutational studies. The activity of hNit2/ω-amidase was determined with α-ketoglutaramate and succinamate as substrates. We constructed three catalytic triad mutants (E43A, K112A, and C153A) and a mutant with a loop 116-128 deletion to validate the role of key residues and the 116-128 loop region in substrate binding and turnover. The molecular dynamics simulations successfully verified the experimental trends in the binding specificity of hNit2/ω-amidase toward various substrates. Our findings have revealed novel structural insights into the binding of substrates to hNit2/ω-amidase. A catalytic triad and the loop residues 116-128 of hNit2 play an essential role in supporting the stability of the enzyme-substrate complex, resulting in the generation of the catalytic products. These observations are predicted to be of benefit in the design of new inhibitors or activators for research involving cancer and hyperammonemic diseases.     

Viper venom hyaluronidase and its potential inhibitor analysis: a multipronged computational investigation

Viper venom hyaluronidase (VV-HYA) inhibitors have long been used as therapeutic agents for arresting the local and systemic effects caused during its envenomation. Henceforth, to understand its structural features and also to identify the best potential inhibitor against it the present computational study was undertaken. Structure-based homology modeling of VV-HYA followed by its docking and free energy-based ranking analysis of ligand, the MD simulations of the lead complex was also performed. The sequence analysis and homology modeling of VV-HYA revealed a distorted (β/α)₈ folding as in the case of hydrolases family of proteins. Molecular docking of the resultant 3D structure of VV-HYA with known inhibitors (compounds 1–25) revealed the importance of molecular recognition of hotspot residues (Tyr 75, Arg 288, and Trp 321) other than that of the active site residues. It also revealed that Trp 321 of VV-HYA is highly important for mediating π–π interactions with ligands. In addition, the molecular docking and comparative free energy binding analysis was investigated for the VV-HYA inhibitors (compounds 1–25). Both molecular docking and relative free energy binding analysis clearly confirmed the identification of sodium chromoglycate (compound 1) as the best potential inhibitor against VV-HYA. Molecular dynamics simulations additionally confirmed the stability of their binding interactions. Further, the information obtained from this work is believed to serve as an impetus for future rational designing of new novel VV-HYA inhibitors with improved activity and selectivity.     

Why is Substrate Peptide Binding Unsusceptible to Multidrug-Resistant Mutations in HIV-1 Protease? A Structural and Energetic Analysis

Understanding of the molecular mechanism and biological implication underlying the difference in binding of substrate peptides and small-molecule inhibitors to multidrug-resistant mutants of HIV-1 protease would help to develop new anti-HIV agents combating drug resistance. Here, an integration of rigorous quantum mechanics/molecular mechanics (QM/MM) analysis and empirical Poisson–Boltzmann/surface area (PB/SA) model is described to investigate the structural basis and energetic property of wild-type HIV-1 protease and its mutants in recognizing and binding with a wide variety of ligands, including the peptides derived from its cognate cleavage sites and the cleavage site variants as well as a number of FDA-approved protease inhibitors, attempting to explain why is substrate binding unsusceptible to most observed HIV-1 protease mutations. A preliminary test study demonstrates that the combined QM/MM–PB/SA scheme is able to effectively reproduce the relative ligand binding energy changes upon protease single- and double-mutations, albeit the absolute values appear to be different significantly between the calculated and experimental results. With the QM/MM–PB/SA calculations a complete mutation energy map of HIV-1 protease–ligand interactions is created, which unravels distinct affinity pictures of wild-type substrates, substrate variants and, particularly, the protease inhibitors bound to HIV-1 protease mutants, suggesting that, on the one hand, the evaluation pressure under anti-HIV chemotherapies addresses site-directed protease mutations that impair and undermine the intermolecular interactions specific to inhibitors but not substrates; on the other hand, co-evaluation of protease and its substrate peptides provides a more effective mechanism to avoid therapeutic surveillance. Further, nonbonded interaction analysis and computational alanine scanning reveal 12 key residues that is critical for substrate binding, from which the Asn25, Gly27, Ala28, Asp29 and Pro81 are identified that have not yet been found to cause drug resistance and hence would be the promising sites targeted by new protease inhibitors.     

Computational simulations of structural role of the active-site W374C mutation of acetyl-coenzyme-A carboxylase: Multi-drug resistance mechanism

Herbicides targeting grass plastidic acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) are selectively effective against graminicides. The intensive worldwide use of this herbicide family has selected for resistance genes in a number of grass weed species. Recently, the active-site W374C mutation was found to confer multi-drug resistance toward haloxyfop (HF), fenoxaprop (FR), Diclofop (DF), and clodinafop (CF) in A. myosuroides. In order to uncover the resistance mechanism due to W374C mutation, the binding of above-mentioned four herbicides to both wild-type and the mutant-type ACCase was investigated in the current work by molecular docking and molecular dynamics (MD) simulations. The binding free energies were calculated by molecular mechanics-Poisson-Boltzmann surface area (MM/PBSA) method. The calculated binding free energy values for four herbicides were qualitatively consistent with the experimental order of IC₅₀ values. All the computational model and energetic results indicated that the W374C mutation has great effects on the conformational change of the binding pocket and the ligand-protein interactions. The most significant conformational change was found to be associated with the aromatic amino acid residues, such as Phe377, Tyr161′ and Trp346. As a result, the π-π interaction between the ligand and the residue of Phe377 and Tyr161′, which make important contributions to the binding affinity, was decreased after mutation and the binding affinity for the inhibitors to the mutant-type ACCase was less than that to the wild-type enzyme, which accounts for the molecular basis of herbicidal resistance. The structural role and mechanistic insights obtained from computational simulations will provide a new starting point for the rational design of novel inhibitors to overcome drug resistance associated with W374C mutation.     

Computational prediction of structure, substrate binding mode, mechanism, and rate for a malaria protease with a novel type of active site

The histo-aspartic protease (HAP) from the malaria parasite P. falciparum is one of several new promising targets for drug intervention. The enzyme possesses a novel type of active site, but its 3D structure and mechanism of action are still unknown. Here we use a combination of homology modeling, automated docking searches, and molecular dynamics/reaction free energy profile simulations to predict the enzyme structure, conformation of bound substrate, catalytic mechanism, and rate of the peptide cleavage reaction. We find that the computational tools are sufficiently reliable both for identifying substrate binding modes and for distinguishing between different possible reaction mechanisms. It is found that the favored pathway only involves direct participation by the catalytic aspartate, with the neighboring histidine providing critical stabilization (by a factor of approximately 10000) along the reaction. The calculated catalytic rate constant of about 0.1 s(-1) for a hexapeptide substrate derived from the alpha chain of human hemoglobin is in excellent agreement with experimental kinetic data for a similar peptide fragment.     

Elucidating substrate and inhibitor binding sites on the surface of GSK-3β and the refinement of a competitive inhibitor

A molecular understanding of substrate recognition of protein kinases provides an important basis for the development of substrate competitive inhibitors. Here, we explored substrate recognition and competitive inhibition of glycogen synthase kinase (GSK)-3β using molecular and computational tools. In previous work, we described Gln89 and Asn95 within GSK-3β as important substrates binding sites. Here, we show that the cavity bordered by loop 89-QDKRFKN-95, located in the vicinity of the GSK-3β catalytic core, is a promiscuous substrate binding subsite. Mutations within this segment highlighted Phe93 as an additional essential contact residue for substrates' recognition. However, unlike Gln89 and Asn95, Phe93 was also important for the binding of our previously described substrate competitive inhibitor, L803 [KEAPPAPPQS(p)P], and its cell-permeable variant L803-mts. The effects of the substitution of charged or polar residues within L803 further suggested that binding to GSK-3β is governed by hydrophobic interactions. Our computational model of GSK-3β bound to L803 was in agreement with the experimental data. It revealed L803 binding with a hydrophobic surface patch and identified interactions between Pro8 (L803) and Phe93 (GSK-3β). Computational modeling of new L803 variants predicted that inhibition would be strengthened by adding contacts with Phe93 or by increasing the hydrophobic content of the peptide. Indeed, the newly designed L803 variants showed improved inhibition. Our study identified different and overlapping elements in GSK-3β substrate and inhibitor recognition and provides a novel example for model-based rational design of substrate competitive inhibitors for GSK-3.     

Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX

Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions.     

Novel missense mutations in gidB gene associated with streptomycin resistance in Mycobacterium tuberculosis: insights from molecular dynamics

Streptomycin was the first antibiotic used for the treatment of tuberculosis by inhibiting translational proof reading. Point mutation in gidB gene encoding S-adenosyl methionine (SAM)-dependent 7-methylguanosine (m7G) methyltransferase required for methylation of 16S rRNA confers streptomycin resistance. As there was no structural substantiation experimentally, gidB protein model was built by threading algorithm. In this work, molecular dynamics (MD) simulations coupled with binding free energy calculations were performed to outline the mechanism underlying high-level streptomycin resistance associated with three novel missense mutants including S70R, T146M, and R187M. Results from dynamics analyses suggested that the structure distortion in the binding pocket of gidB mutants modulate SAM binding affinity. At the structural level, these conformational changes bring substantial decrease in the number of residues involved in hydrogen bonding and dramatically reduce thermodynamic stability of mutant gidB–SAM complexes. The outcome of comparative analysis of the MD simulation trajectories revealed lower conformational stability associated with higher flexibility in mutants relative to the wild-type, turns to be major factor driving the emergence of drug resistance toward antibiotic. This study will pave way toward design and development of resistant defiant gidB inhibitors as potent anti-TB agents.     

An Induced Pocket for the Binding of Potent Fusion Inhibitor CL-385319 with H5N1 Influenza Virus Hemagglutinin

The influenza glycoprotein hemagglutinin (HA) plays crucial roles in the early stage of virus infection, including receptor binding and membrane fusion. Therefore, HA is a potential target for developing anti-influenza drugs. Recently, we characterized a novel inhibitor of highly pathogenic H5N1 influenza virus, CL-385319, which specifically inhibits HA-mediated viral entry. Studies presented here identified the critical binding residues for CL-385319, which clustered in the stem region of the HA trimer by site-directed mutagenesis. Extensive computational simulations, including molecular docking, molecular dynamics simulations, molecular mechanics generalized Born surface area (MM_GBSA) calculations, charge density and Laplacian calculations, have been carried out to uncover the detailed molecular mechanism that underlies the binding of CL-385319 to H5N1 influenza virus HA. It was found that the recognition and binding of CL-385319 to HA proceeds by a process of "induced fit" whereby the binding pocket is formed during their interaction. Occupation of this pocket by CL-385319 stabilizes the neutral pH structure of hemagglutinin, thus inhibiting the conformational rearrangements required for membrane fusion. This "induced fit" pocket may be a target for structure-based design of more potent influenza fusion inhibitors.     

Dual anti-inflammatory and selective inhibition mechanism of leukotriene A4 hydrolase/aminopeptidase: insights from comparative molecular dynamics and binding free energy analyses

Human leukotriene A₄ hydrolase/aminopeptidase (LTA₄H) is a zinc metalloenzyme with a dual catalytic activity; conversion of LTA₄ into LTB₄ and degradation of chemotactic tripeptide Pro-Gly-Pro (PGP). Existing inhibitors, such as SC-57461A, block both catalytic activities of the enzyme, leading to drug failures. Recently, a novel compound, ARM1, was reported to selectively inhibit the hydrolase activity of LTA₄H while sparing its aminopeptidase activity. However, the molecular understanding of such preferential inhibitory mechanism remains obscure. The discovery of ARM1 prompted us to further explore its binding theme and provide more insight into the structural and dual mechanistic features of LTA₄H protein. To accomplish this, we embarked on wide range of computational tools, including comparative molecular dynamics (MDs) simulations and postdynamic analyses for LTA₄H and in complex with ARM1, PGP, ARM1-PGP, and SC-57461A. MD analysis reveals that the binding of ARM1 exhibits a more stable active site and overall stable protein conformation when compared to the nonselective inhibitor SC-57461A. In addition, MM/GBSA-binding free energy calculation also reveals that ARM1 exhibit a lower binding affinity, when compared to the nonselective inhibitor SC-57461A – which is in a great agreement with experimental data. Per residue energy decomposition analysis showed that Phe314, Val367, Tyr378, Trp311, Pro382, and Leu369 are key residues critical for the selective inhibition of the epoxide hydrolase activity of LTA₄H by ARM1. Findings from this report will not only provide more understanding into the structural, dynamic, and mechanistic features of LTA₄H but would also assist toward the rational design of novel and selective hydrolase inhibitors of LTA₄H as anti-inflammatory drugs.     

Probing the structural and energetic basis of kinesin-microtubule binding using computational alanine-scanning mutagenesis

Kinesin-microtubule (MT) binding plays a critical role in facilitating and regulating the motor function of kinesins. To obtain a detailed structural and energetic picture of kinesin-MT binding, we performed large-scale computational alanine-scanning mutagenesis based on long-time molecular dynamics (MD) simulations of the kinesin-MT complex in both ADP and ATP states. First, we built three all-atom kinesin-MT models: human conventional kinesin bound to ADP and mouse KIF1A bound to ADP and ATP. Then, we performed 30 ns MD simulations followed by kinesin-MT binding free energy calculations for both the wild type and mutants obtained after substitution of each charged residue of kinesin with alanine. We found that the kinesin-MT binding free energy is dominated by van der Waals interactions and further enhanced by electrostatic interactions. The calculated mutational changes in kinesin-MT binding free energy are in excellent agreement with results of an experimental alanine-scanning study with a root-mean-square error of ~0.32 kcal/mol [Woehlke, G., et al. (1997) Cell 90, 207-216]. We identified a set of important charged residues involved in the tuning of kinesin-MT binding, which are clustered on several secondary structural elements of kinesin (including well-studied loops L7, L8, L11, and L12, helices α4, α5, and α6, and less-explored loop L2). In particular, we found several key residues that make different contributions to kinesin-MT binding in ADP and ATP states. The mutations of these residues are predicted to fine-tune the motility of kinesin by modulating the conformational transition between the ADP state and the ATP state of kinesin.