Asymmetric framework motion of TCRαß controls load-dependent peptide discrimination.

Mechanical force is critical for the interaction between an αß T cell receptor (TCR) and a peptide-bound major histocompatibility complex (pMHC) molecule to initiate productive T-cell activation. However, the underlying mechanism remains unclear. We use all-atom molecular dynamics simulations to examine the A6 TCR bound to HLA-A*02:01 presenting agonist or antagonist peptides under different extensions to simulate the effects of applied load on the complex, elucidating their divergent biological responses. We found that TCR α and ß chains move asymmetrically, which impacts the interface with pMHC, in particular the peptide-sensing CDR3 loops. For the wild-type agonist, the complex stabilizes in a load-dependent manner while antagonists destabilize it. Simulations of the Cß FG-loop deletion, which reduces the catch bond response, and simulations with in silico mutant peptides further support the observed behaviors. The present results highlight the combined role of interdomain motion, fluctuating forces, and interfacial contacts in determining the mechanical response and fine peptide discrimination by a TCR, thereby resolving the conundrum of nearly identical crystal structures of TCRαß-pMHC agonist and antagonist complexes.

Parsing digital or analogue TCR performance through piconewton forces.

αß T-cell receptors (TCRs) recognize aberrant peptides bound to major histocompatibility complex molecules (pMHCs) on unhealthy cells, amplifying specificity and sensitivity through physical load placed on the TCR-pMHC bond during immunosurveillance. To understand this mechanobiology, TCRs stimulated by abundantly and sparsely arrayed epitopes (NP 366-374 /D b and PA 224-233 /D b , respectively) following in vivo influenza A virus infection were studied with optical tweezers. While certain NP repertoire CD8 T lymphocytes require many ligands for activation, others are digital, needing just few. Conversely, all PA TCRs perform digitally, exhibiting pronounced bond lifetime increases through sustained, energizing volleys of structural transitioning. Optimal digital performance is superior in vivo, correlating with ERK phosphorylation, CD3 loss, and activation marker upregulation in vitro . Given neoantigen array paucity, digital TCRs are likely critical for immunotherapies. One Sentence Summary: Quality of ligand recognition in a T-cell repertoire is revealed through application of physical load on clonal T-cell receptor (TCR)-pMHC bonds.

Inadequate structural constraint on Fab approach rather than paratope elicitation limits HIV-1 MPER vaccine utility.

Broadly neutralizing antibodies (bnAbs) against HIV-1 target conserved envelope (Env) epitopes to block viral replication. Here, using structural analyses, we provide evidence to explain why a vaccine targeting the membrane-proximal external region (MPER) of HIV-1 elicits antibodies with human bnAb-like paratopes paradoxically unable to bind HIV-1. Unlike in natural infection, vaccination with MPER/liposomes lacks a necessary structure-based constraint to select for antibodies with an adequate approach angle. Consequently, the resulting Abs cannot physically access the MPER crawlspace on the virion surface. By studying naturally arising Abs, we further reveal that flexibility of the human IgG3 hinge mitigates the epitope inaccessibility and additionally facilitates Env spike protein crosslinking. Our results suggest that generation of IgG3 subtype class-switched B cells is a strategy for anti-MPER bnAb induction. Moreover, the findings illustrate the need to incorporate topological features of the target epitope in immunogen design.

Asymmetric framework motion of TCRαß controls load-dependent peptide discrimination.

Mechanical force is critical for the interaction between an αßT cell receptor (TCR) and a peptide-bound major histocompatibility complex (pMHC) molecule to initiate productive T-cell activation. However, the underlying mechanism remains unclear. We use all-atom molecular dynamics simulations to examine the A6 TCR bound to HLA-A*02:01 presenting agonist or antagonist peptides under different extensions to simulate the effects of applied load on the complex, elucidating their divergent biological responses. We found that TCR α and ß chains move asymmetrically, which impacts the interface with pMHC, in particular the peptide-sensing CDR3 loops. For the wild-type agonist, the complex stabilizes in a load-dependent manner while antagonists destabilize it. Simulations of the Cß FG-loop deletion, which reduces the catch bond response, and simulations with in silico mutant peptides further support the observed behaviors. The present results highlight the combined role of interdomain motion, fluctuating forces, and interfacial contacts in determining the mechanical response and fine peptide discrimination by a TCR, thereby resolving the conundrum of nearly identical crystal structures of TCRαß-pMHC agonist and antagonist complexes.

Inadequate structural constraint on Fab approach rather than paratope elicitation limits HIV-1 MPER vaccine utility.

Broadly neutralizing antibodies (bnAbs) against HIV-1 target conserved epitopes, thereby inhibiting viral entry. Yet surprisingly, those recognizing linear epitopes in the HIV-1 gp41 membrane proximal external region (MPER) are elicited neither by peptide nor protein scaffold vaccines. Here, we observe that while Abs generated by MPER/liposome vaccines may exhibit human bnAb-like paratopes, B-cell programming without constraints imposed by the gp160 ectodomain selects Abs unable to access the MPER within its native "crawlspace". During natural infection, the flexible hinge of IgG3 partially mitigates steric occlusion of less pliable IgG1 subclass Abs with identical MPER specificity, until affinity maturation refines entry mechanisms. The IgG3 subclass maintains B-cell competitiveness, exploiting bivalent ligation resulting from greater intramolecular Fab arm length, offsetting weak antibody affinity. These findings suggest future immunization strategies.

Harnessing αß T cell receptor mechanobiology to achieve the promise of immuno-oncology.

T cell receptors (TCR) on cytolytic T lymphocytes (CTLs) recognize "foreign" antigens bound in the groove of major histocompatibility complex (MHC) molecules (H-2 in mouse and HLA in human) displayed on altered cells. These antigens are peptide fragments of proteins derived either from infectious pathogens or cellular transformations during cancer evolution. The conjoint ligand formed by the foreign peptide and MHC, termed pMHC, marks an aberrant cell as a target for CTL-mediated destruction. Recent data have provided compelling evidence that adaptive protection is achieved in a facile manner during immune surveillance when mechanical load consequent to cellular motion is applied to the bond formed between an αß TCR and its pMHC ligand arrayed on a disease-altered cell. Mechanobiology maximizes both TCR specificity and sensitivity in comparison to receptor ligation in the absence of force. While the field of immunotherapy has made advances to impact the survival of cancer patients, the latest information relevant to T cell targeting and mechanotransduction has yet to be applied for T cell monitoring and treatment of patients in the clinic. Here we review these data, and challenge scientists and physicians to apply critical biophysical parameters of TCR mechanobiology to the medical oncology field, broadening treatment success within and among various cancer types. We assert that TCRs with digital ligand-sensing performance capability directed at sparsely as well as luminously displayed tumor-specific neoantigens and certain tumor-associated antigens can improve effective cancer vaccine development and immunotherapy paradigms.

Heterogeneous and Allosteric Role of Surface Hydration for Protein-Ligand Binding.

Atomistic-level understanding of surface hydration mediating protein-protein interactions and ligand binding has been a challenge due to the dynamic nature of water molecules near the surface. We develop a computational method to evaluate the solvation free energy based on the density map of the first hydration shell constructed from all-atom molecular dynamics simulation and use it to examine the binding of two intrinsically disordered ligands to their target protein domain. One ligand is from the human protein, and the other is from the 1918 Spanish flu virus. We find that the viral ligand incurs a 6.9 kcal/mol lower desolvation penalty upon binding to the target, which is consistent with its stronger binding affinity. The difference arises from the spatially fragmented and nonuniform water density profiles of the first hydration shell. In particular, residues that are distal from the ligand-binding site contribute to a varying extent to the desolvation penalty, among which the "entropy hotspot" residues contribute significantly. Thus, ligand binding alters hydration on remote sites in addition to affecting the binding interface. The nonlocal effect disappears when the conformational motion of the protein is suppressed. The present results elucidate the interplay between protein conformational dynamics and surface hydration. Our approach of measuring the solvation free energy based on the water density of the first hydration shell is tolerant of the conformational fluctuation of protein, and we expect it to be applicable to investigating a broad range of biomolecular interfaces.

Pre-T cell receptor self-MHC sampling restricts thymocyte dedifferentiation.

Programming T cells to distinguish self from non-self is a vital, multi-step process that occurs in the thymus1-4. Signalling through the pre-T cell receptor (preTCR), a CD3-associated heterodimer comprising an invariant pTα chain and a clone-specific ß chain, is a critical early checkpoint in thymocyte development within the αß T cell lineage5,6. PreTCRs arrayed on CD4-CD8- double-negative thymocytes ligate peptides bound to major histocompatibility complex molecules (pMHC) on thymic stroma, similar to αß T cell receptors that appear on CD4+CD8+ double-positive thymocytes, but via a different molecular docking strategy7-10. Here we show the consequences of these distinct interactions for thymocyte progression using synchronized fetal thymic progenitor cultures that differ in the presence or absence of pMHC on support stroma, and single-cell transcriptomes at key thymocyte developmental transitions. Although major histocompatibility complex (MHC)-negative stroma fosters αß T cell differentiation, the absence of preTCR-pMHC interactions leads to deviant thymocyte transcriptional programming associated with dedifferentiation. Highly proliferative double-negative and double-positive thymocyte subsets emerge, with antecedent characteristics of T cell lymphoblastic and myeloid malignancies. Compensatory upregulation of diverse MHC class Ib proteins in B2m/H2-Ab1 MHC-knockout mice partially safeguards in vivo thymocyte progression, although disseminated double-positive thymic tumours may develop with ageing. Thus, as well as promoting ß chain repertoire broadening for subsequent αß T cell receptor utilization, preTCR-pMHC interactions limit cellular plasticity to facilitate normal thymocyte differentiation and proliferation that, if absent, introduce developmental vulnerabilities.

Building a three-dimensional model of early-stage zebrafish embryo brain.

We introduce a computational approach to build three-dimensional (3D) surface mesh models of the early-stage zebrafish brain primordia from time-series microscopy images. The complexity of the early-stage brain primordia and lack of recognizable landmarks pose a distinct challenge for feature segmentation and 3D modeling. Additional difficulty arises because of noise and variations in pixel intensity. We overcome these by using a hierarchical approach in which simple geometric elements, such as "beads" and "bonds," are assigned to represent local features and their connectivity is used to smoothen the surface while retaining high-curvature regions. We apply our method to build models of two zebrafish embryo phenotypes at discrete time points between 19 and 28 h post-fertilization and collect measurements to quantify development. Our approach is fast and applicable to building models of other biological systems, as demonstrated by models from magnetic resonance images of the human fetal brain. The source code, input scripts, sample image files, and generated outputs are publicly available on GitHub.

Molecular design of the γδT cell receptor ectodomain encodes biologically fit ligand recognition in the absence of mechanosensing.

High-acuity αßT cell receptor (TCR) recognition of peptides bound to major histocompatibility complex molecules (pMHCs) requires mechanosensing, a process whereby piconewton (pN) bioforces exert physical load on αßTCR-pMHC bonds to dynamically alter their lifetimes and foster digital sensitivity cellular signaling. While mechanotransduction is operative for both αßTCRs and pre-TCRs within the αßT lineage, its role in γδT cells is unknown. Here, we show that the human DP10.7 γδTCR specific for the sulfoglycolipid sulfatide bound to CD1d only sustains a significant load and undergoes force-induced structural transitions when the binding interface-distal γδ constant domain (C) module is replaced with that of αß. The chimeric γδ-αßTCR also signals more robustly than does the wild-type (WT) γδTCR, as revealed by RNA-sequencing (RNA-seq) analysis of TCR-transduced Rag2-/- thymocytes, consistent with structural, single-molecule, and molecular dynamics studies reflective of γδTCRs as mediating recognition via a more canonical immunoglobulin-like receptor interaction. Absence of robust, force-related catch bonds, as well as γδTCR structural transitions, implies that γδT cells do not use mechanosensing for ligand recognition. This distinction is consonant with the fact that their innate-type ligands, including markers of cellular stress, are expressed at a high copy number relative to the sparse pMHC ligands of αßT cells arrayed on activating target cells. We posit that mechanosensing emerged over ∼200 million years of vertebrate evolution to fulfill indispensable adaptive immune recognition requirements for pMHC in the αßT cell lineage that are unnecessary for the γδT cell lineage mechanism of non-pMHC ligand detection.

Pre-T cell receptors topologically sample self-ligands during thymocyte ß-selection.

Self-discrimination, a critical but ill-defined molecular process programmed during thymocyte development, requires myriad pre-T cell receptors (preTCRs) and αßTCRs. Using x-ray crystallography, we show how a preTCR applies the concave ß-sheet surface of its single variable domain (Vß) to "horizontally" grab the protruding MHC α2-helix. By contrast, αßTCRs purpose all six complementarity-determining region (CDR) loops of their paired VαVß module to recognize peptides bound to major histocompatibility complex molecules (pMHCs) in "vertical" head-to-head binding. The preTCR topological fit ensures that CDR3ß reaches the peptide's featured C-terminal segment for pMHC sampling, establishing the subsequent αßTCR canonical docking mode. "Horizontal" docking precludes germline CDR1ß- and CDR2ß-MHC binding to broaden ß-chain repertoire diversification before αßTCR-mediated selection refinement. Thus, one subunit successively attunes the recognition logic of related multicomponent receptors.

The αßTCR mechanosensor exploits dynamic ectodomain allostery to optimize its ligand recognition site.

Each [Formula: see text]T cell receptor (TCR) functions as a mechanosensor. The TCR is comprised of a clonotypic TCR[Formula: see text] ligand-binding heterodimer and the noncovalently associated CD3 signaling subunits. When bound by ligand, an antigenic peptide arrayed by a major histocompatibility complex molecule (pMHC), the TCR[Formula: see text] has a longer bond lifetime under piconewton-level loads. The atomistic mechanism of this "catch bond" behavior is unknown. Here, we perform molecular dynamics simulation of a TCR[Formula: see text]-pMHC complex and its variants under physiologic loads to identify this mechanism and any attendant TCR[Formula: see text] domain allostery. The TCR[Formula: see text]-pMHC interface is dynamically maintained by contacts with a spectrum of occupancies, introducing a level of control via relative motion between Vα and Vß variable domains containing the pMHC-binding complementarity-determining region (CDR) loops. Without adequate load, the interfacial contacts are unstable, whereas applying sufficient load suppresses Vα-Vß motion, stabilizing the interface. A second level of control is exerted by Cα and Cß constant domains, especially Cß and its protruding FG-loop, that create mismatching interfaces among the four TCR[Formula: see text] domains and with a pMHC ligand. Applied load enhances fit through deformation of the TCR[Formula: see text] molecule. Thus, the catch bond involves the entire TCR[Formula: see text] conformation, interdomain motion, and interfacial contact dynamics, collectively. This multilayered architecture of the machinery fosters fine-tuning of cellular response to load and pMHC recognition. Since the germline-derived TCR[Formula: see text] ectodomain is structurally conserved, the proposed mechanism can be universally adopted to operate under load during immune surveillance by diverse [Formula: see text]TCRs constituting the T cell repertoire.

Entropy Hotspots for the Binding of Intrinsically Disordered Ligands to a Receptor Domain.

Proline-rich motifs (PRMs) are widely used for mediating protein-protein interactions with weak binding affinities. Because they are intrinsically disordered when unbound, conformational entropy plays a significant role for the binding. However, residue-level differences of the entropic contribution in the binding of different ligands remain not well understood. We use all-atom molecular dynamics simulation and the maximal information spanning tree formalism to analyze conformational entropy associated with the binding of two PRMs, one from the Abl kinase and the other from the nonstructural protein 1 of the 1918 Spanish influenza A virus, to the N-terminal SH3 (nSH3) domain of the CrkII protein. Side chains of the stably folded nSH3 experience more entropy change upon ligand binding than the backbone, whereas PRMs involve comparable but heterogeneous entropy changes among the backbone and side chains. In nSH3, two conserved nonpolar residues forming contacts with the PRM experience the largest side-chain entropy loss. In contrast, the C-terminal charged residues of PRMs that form polar contacts with nSH3 experience the greatest side-chain entropy loss, although their "fuzzy" nature is attributable to the backbone that remains relatively flexible. Thus, residues that form high-occupancy contacts between nSH3 and PRM do not reciprocally contribute to entropy loss. Furthermore, certain surface residues of nSH3 distal to the interface with PRMs gain entropy, indicating a nonlocal effect of ligand binding. Comparing between the PRMs from cAbl and nonstructural protein 1, the latter involves a larger side-chain entropy loss and forms more contacts with nSH3. Consistent with experiments, this indicates stronger binding of the viral ligand at the expense of losing the flexibility of side chains, whereas the backbone experiences less entropy loss. The entropy "hotspots" as identified in this study will be important for tuning the binding affinity of various ligands to a receptor.

Molecular recognition of a host protein by NS1 of pandemic and seasonal influenza A viruses.

The 1918 influenza A virus (IAV) caused the most severe flu pandemic in recorded human history. Nonstructural protein 1 (NS1) is an important virulence factor of the 1918 IAV. NS1 antagonizes host defense mechanisms through interactions with multiple host factors. One pathway by which NS1 increases virulence is through the activation of phosphoinositide 3-kinase (PI3K) by binding to its p85ß subunit. Here we present the mechanism underlying the molecular recognition of the p85ß subunit by 1918 NS1. Using X-ray crystallography, we determine the structure of 1918 NS1 complexed with p85ß of human PI3K. We find that the 1918 NS1 effector domain (1918 NS1ED) undergoes a conformational change to bind p85ß. Using NMR relaxation dispersion and molecular dynamics simulation, we identify that free 1918 NS1ED exists in a dynamic equilibrium between p85ß-binding-competent and -incompetent conformations in the submillisecond timescale. Moreover, we discover that NS1ED proteins of 1918 (H1N1) and Udorn (H3N2) strains exhibit drastically different conformational dynamics and binding kinetics to p85ß. These results provide evidence of strain-dependent conformational dynamics of NS1. Using kinetic modeling based on the experimental data, we demonstrate that 1918 NS1ED can result in the faster hijacking of p85ß compared to Ud NS1ED, although the former has a lower affinity to p85ß than the latter. Our results suggest that the difference in binding kinetics may impact the competition with cellular antiviral responses for the activation of PI3K. We anticipate that our findings will increase the understanding of the strain-dependent behaviors of influenza NS1 proteins.

Structural basis for power stroke vs. Brownian ratchet mechanisms of motor proteins.

Two mechanisms have been proposed for the function of motor proteins: The power stroke and the Brownian ratchet. The former refers to generation of a large downhill free energy gradient over which the motor protein moves nearly irreversibly in making a step, whereas the latter refers to biasing or rectifying the diffusive motion of the motor. Both mechanisms require input of free energy, which generally involves the processing of an ATP (adenosine 5'-triphosphate) molecule. Recent advances in experiments that reveal the details of the stepping motion of motor proteins, together with computer simulations of atomistic structures, have provided greater insights into the mechanisms. Here, we compare the various models of the power stroke and the Brownian ratchet that have been proposed. The 2 mechanisms are not mutually exclusive, and various motor proteins employ them to different extents to perform their biological function. As examples, we discuss linear motor proteins Kinesin-1 and myosin-V, and the rotary motor F1-ATPase, all of which involve a power stroke as the essential element of their stepping mechanism.

Role of mechanical flow for actin network organization.

The major cytoskeletal protein actin forms complex networks to provide structural support and perform vital functions in cells. In vitro studies have revealed that the structure of the higher-order actin network is determined primarily by the type of actin binding protein (ABP). By comparison, there are far fewer studies about the role of the mechanical environment for the organization of the actin network. In particular, the duration over which cells reorganize their shape in response to functional demands is relatively short compared to the in vitro protein polymerization time, suggesting that such changes can influence the actin network formation. We hypothesize that mechanical flows in the cytoplasm generated by exogenous and endogenous stimulation play a key role in the spatiotemporal regulation of the actin architecture. To mimic cytoplasmic streaming, we generated a circulating flow using surface acoustic wave in a microfluidic channel and investigated its effect on the formation of networks by actin and ABPs. We found that the mechanical flow affected the orientation and thickness of actin bundles, depending on the type and concentration of ABPs. Our computational model shows that the extent of alignment and thickness of actin bundle are determined by the balance between flow-induced drag forces and the tendency of ABPs to crosslink actin filaments at given angles. These results suggest that local intracellular flows can affect the assembly dynamics and morphology of the actin cytoskeleton. STATEMENT OF SIGNIFICANCE: Spatiotemporal regulation of actin cytoskeleton structure is essential in many cellular functions. It has been shown that mechanical cues including an applied force and geometric boundary can alter the structural characteristics of actin network. However, even though the cytoplasm accounts for a large portion of the cell volume, the effect of the cytoplasmic streaming flow produced during cell dynamics on actin network organization has not been reported. In this study, we demonstrated that the mechanical flow exerted during actin network organization play an important role in determining the orientation and dimension of actin bundle network. Our result will be beneficial in understanding the mechanism of the actin network reorganization occurred during physiological and pathological processes.

NMR: an essential structural tool for integrative studies of T cell development, pMHC ligand recognition and TCR mechanobiology.

Early studies of T cell structural biology using X-ray crystallography, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) focused on a picture of the αßT cell receptor (αßTCR) component domains and their cognate ligands (peptides bound to MHC molecules, i.e. pMHCs) as static interaction partners. Moving forward requires integrating this corpus of data with dynamic technologies such as NMR, molecular dynamics (MD) simulations and real-time single molecule (SM) studies exemplified by optical tweezers (OT). NMR bridges relevant timescales and provides the potential for an all-atom dynamic description of αßTCR components prior to and during interactions with binding partners. SM techniques have opened up vistas in understanding the non-equilibrium nature of T cell signaling through the introduction of force-mediated binding measurements into the paradigm for T cell function. In this regard, bioforces consequent to T-lineage cell motility are now perceived as placing piconewton (pN)-level loads on single receptor-pMHC bonds to impact structural change and αßT-lineage biology, including peptide discrimination, cellular activation, and developmental progression. We discuss herein essential NMR technologies in illuminating the role of ligand binding in the preT cell receptor (preTCR), the αßTCR developmental precursor, and convergence of NMR, SM and MD data in advancing our comprehension of T cell development. More broadly we review the central hypothesis that the αßTCR is a mechanosensor, fostered by breakthrough NMR-based structural insights. Collectively, elucidating dynamic aspects through the integrative use of NMR, SM, and MD shall advance fundamental appreciation of the mechanism of T cell signaling as well as inform translational efforts in αßTCR and chimeric T cell (CAR-T) immunotherapies and T cell vaccinology.

Effect of Methylation on Local Mechanics and Hydration Structure of DNA.

Cytosine methylation affects mechanical properties of DNA and potentially alters the hydration fingerprint for recognition by proteins. The atomistic origin for these effects is not well understood, and we address this via all-atom molecular dynamics simulations. We find that the stiffness of the methylated dinucleotide step changes marginally, whereas the neighboring steps become stiffer. Stiffening is further enhanced for consecutively methylated steps, providing a mechanistic origin for the effect of hypermethylation. Steric interactions between the added methyl groups and the nonpolar groups of the neighboring nucleotides are responsible for the stiffening in most cases. By constructing hydration maps, we found that methylation also alters the surface hydration structure in distinct ways. Its resistance to deformation may contribute to the stiffening of DNA for deformational modes lacking steric interactions. These results highlight the sequence- and deformational-mode-dependent effects of cytosine methylation.

Molecular Mechanisms of Tight Binding through Fuzzy Interactions.

Many intrinsically disordered proteins (IDPs) form fuzzy complexes upon binding to their targets. Although many IDPs are weakly bound in fuzzy complexes, some IDPs form high-affinity complexes. One example is the nonstructural protein 1 (NS1) of the 1918 Spanish influenza A virus, which hijacks cellular CRKII through the strong binding affinity (Kd ∼10 nM) of its proline-rich motif (PRMNS1) to the N-terminal Src-homology 3 domain of CRKII. However, its molecular mechanism remains elusive. Here, we examine the interplay between structural disorder of a bound PRMNS1 and its long-range electrostatic interactions. Using x-ray crystallography and NMR spectroscopy, we found that PRMNS1 retains substantial conformational flexibility in the bound state. Moreover, molecular dynamics simulations showed that structural disorder of the bound PRMNS1 increases the number of electrostatic interactions and decreases the mean distances between the positively charged residues in PRMNS1 and the acidic residues in the N-terminal Src-homology 3 domain. These results are analyzed using a polyelectrostatic model. Our results provide an insight into the molecular recognition mechanism for a high-affinity fuzzy complex.

Kinesin motility is driven by subdomain dynamics.

The microtubule (MT)-associated motor protein kinesin utilizes its conserved ATPase head to achieve diverse motility characteristics. Despite considerable knowledge about how its ATPase activity and MT binding are coupled to the motility cycle, the atomic mechanism of the core events remain to be found. To obtain insights into the mechanism, we performed 38.5 microseconds of all-atom molecular dynamics simulations of kinesin-MT complexes in different nucleotide states. Local subdomain dynamics were found to be essential for nucleotide processing. Catalytic water molecules are dynamically organized by the switch domains of the nucleotide binding pocket while ATP is torsionally strained. Hydrolysis products are 'pulled' by switch-I, and a new ATP is 'captured' by a concerted motion of the α0/L5/switch-I trio. The dynamic and wet kinesin-MT interface is tuned for rapid interactions while maintaining specificity. The proposed mechanism provides the flexibility necessary for walking in the crowded cellular environment.