PCMRESP: A Method for Polarizable Force Field Parameter Development and Transferability of the Polarizable Gaussian Multipole Models Across Multiple Solvents.

The transferability of force field parameters is a crucial aspect of high-quality force fields. Previous investigations have affirmed the transferability of electrostatic parameters derived from polarizable Gaussian multipole models (pGMs) when applied to water oligomer clusters, polypeptides across various conformations, and different sequences. In this study, we introduce PCMRESP, a novel method for electrostatic parametrization in solution, intended for the development of polarizable force fields. We utilized this method to assess the transferability of three models: a fixed charge model and two variants of pGM models. Our analysis involved testing these models on 377 small molecules and 100 tetra-peptides in five representative dielectric environments: gas, diethyl ether, dichloroethane, acetone, and water. Our findings reveal that the inclusion of atomic polarization significantly enhances transferability and the incorporation of permanent atomic dipoles, in the form of covalent bond dipoles, leads to further improvements. Moreover, our tests on dual-solvent strategies demonstrate consistent transferability for all three models, underscoring the robustness of the dual-solvent approach. In contrast, an evaluation of the traditional HF/6-31G* method indicates poor transferability for the pGM-ind and pGM-perm models, suggesting the limitations of this conventional approach.

Assessment of Amino Acid Electrostatic Parametrizations of the Polarizable Gaussian Multipole Model.

Accurate parametrization of amino acids is pivotal for the development of reliable force fields for molecular modeling of biomolecules such as proteins. This study aims to assess amino acid electrostatic parametrizations with the polarizable Gaussian Multipole (pGM) model by evaluating the performance of the pGM-perm (with atomic permanent dipoles) and pGM-ind (without atomic permanent dipoles) variants compared to the traditional RESP model. The 100-conf-combterm fitting strategy on tetrapeptides was adopted, in which (1) all peptide bond atoms (-CO-NH-) share identical set of parameters and (2) the total charges of the two terminal N-acetyl (ACE) and N-methylamide (NME) groups were set to neutral. The accuracy and transferability of electrostatic parameters across peptides with varying lengths and real-world examples were examined. The results demonstrate the enhanced performance of the pGM-perm model in accurately representing the electrostatic properties of amino acids. This insight underscores the potential of the pGM-perm model and the 100-conf-combterm strategy for the future development of the pGM force field.

Dual function of Zika virus NS2B-NS3 protease.

Zika virus (ZIKV) serine protease, indispensable for viral polyprotein processing and replication, is composed of the membrane-anchored NS2B polypeptide and the N-terminal domain of the NS3 polypeptide (NS3pro). The C-terminal domain of the NS3 polypeptide (NS3hel) is necessary for helicase activity and contains an ATP-binding site. We discovered that ZIKV NS2B-NS3pro binds single-stranded RNA with a Kd of ~0.3 µM, suggesting a novel function. We tested various structural modifications of NS2B-NS3pro and observed that constructs stabilized in the recently discovered "super-open" conformation do not bind RNA. Likewise, stabilizing NS2B-NS3pro in the "closed" (proteolytically active) conformation using substrate inhibitors abolished RNA binding. We posit that RNA binding occurs when ZIKV NS2B-NS3pro adopts the "open" conformation, which we modeled using highly homologous dengue NS2B-NS3pro crystallized in the open conformation. We identified two positively charged fork-like structures present only in the open conformation of NS3pro. These forks are conserved across Flaviviridae family and could be aligned with the positively charged grove on NS3hel, providing a contiguous binding surface for the negative RNA strand exiting helicase. We propose a "reverse inchworm" model for a tightly intertwined NS2B-NS3 helicase-protease machinery, which suggests that NS2B-NS3pro cycles between open and super-open conformations to bind and release RNA enabling long-range NS3hel processivity. The transition to the closed conformation, likely induced by the substrate, enables the classical protease activity of NS2B-NS3pro.

Streamlining and Optimizing Strategies of Electrostatic Parameterization.

Accurate characterization of electrostatic interactions is crucial in molecular simulation. Various methods and programs have been developed to obtain electrostatic parameters for additive or polarizable models to replicate electrostatic properties obtained from experimental measurements or theoretical calculations. Electrostatic potentials (ESPs), a set of physically well-defined observables from quantum mechanical (QM) calculations, are well suited for optimization efforts due to the ease of collecting a large amount of conformation-dependent data. However, a reliable set of QM ESP computed at an appropriate level of theory and atomic basis set is necessary. In addition, despite the recent development of the PyRESP program for electrostatic parameterizations of induced dipole-polarizable models, the time-consuming and error-prone input file preparation process has limited the widespread use of these protocols. This work aims to comprehensively evaluate the quality of QM ESPs derived by eight methods, including wave function methods such as Hartree-Fock (HF), second-order Møller-Plesset (MP2), and coupled cluster-singles and doubles (CCSD), as well as five hybrid density functional theory (DFT) methods, used in conjunction with 13 different basis sets. The highest theory levels CCSD/aug-cc-pV5Z (a5z) and MP2/aug-cc-pV5Z (a5z) were selected as benchmark data over two homemade data sets. The results show that the hybrid DFT method, ωB97X-D, combined with the aug-cc-pVTZ (a3z) basis set, performs well in reproducing ESPs while taking both accuracy and efficiency into consideration. Moreover, a flexible and user-friendly program called PyRESP_GEN was developed to streamline input file preparation. The restraining strengths, along with strategies for polarizable Gaussian multipole (pGM) model parameterizations, were also optimized. These findings and the program presented in this work facilitate the development and application of induced dipole-polarizable models, such as pGM models, for molecular simulations of both chemical and biological significance.

Optimal Scheme to Achieve Energy Conservation in Induced Dipole Models.

Induced dipole models have proven to be effective tools for simulating electronic polarization effects in biochemical processes, yet their potential has been constrained by energy conservation issue, particularly when historical data is utilized for dipole prediction. This study identifies error outliers as the primary factor causing this failure of energy conservation and proposes a comprehensive scheme to overcome this limitation. Leveraging maximum relative errors as a convergence metric, our data demonstrates that energy conservation can be upheld even when using historical information for dipole predictions. Our study introduces the multi-order extrapolation method to quicken induction iteration and optimize the use of historical data, while also developing the preconditioned conjugate gradient with local iterations to refine the iteration process and effectively remove error outliers. This scheme further incorporates a "peek" step via Jacobi under-relaxation for optimal performance. Simulation evidence suggests that our proposed scheme can achieve energy convergence akin to that of point-charge models within a limited number of iterations, thus promising significant improvements in efficiency and accuracy.

Transferability of the Electrostatic Parameters of the Polarizable Gaussian Multipole Model.

Accuracy and transferability are the two highly desirable properties of molecular mechanical force fields. Compared with the extensively used point-charge additive force fields that apply fixed atom-centered point partial charges to model electrostatic interactions, polarizable force fields are thought to have the advantage of modeling the atomic polarization effects. Previous works have demonstrated the accuracy of the recently developed polarizable Gaussian multipole (pGM) models. In this work, we assessed the transferability of the electrostatic parameters of the pGM models with (pGM-perm) and without (pGM-ind) atomic permanent dipoles in terms of reproducing the electrostatic potentials surrounding molecules/oligomers absent from electrostatic parameterizations. Encouragingly, both the pGM-perm and pGM-ind models show significantly improved transferability than the additive model in the tests (1) from water monomer to water oligomer clusters; (2) across different conformations of amino acid dipeptides and tetrapeptides; (3) from amino acid tetrapeptides to longer polypeptides; and (4) from nucleobase monomers to Watson-Crick base pair dimers and tetramers. Furthermore, we demonstrated that the double-conformation fittings using amino acid tetrapeptides in the αR and ß conformations can result in good transferability not only across different tetrapeptide conformations but also from tetrapeptides to polypeptides with lengths ranging from 1 to 20 repetitive residues for both the pGM-ind and pGM-perm models. In addition, the observation that the pGM-ind model has significantly better accuracy and transferability than the point-charge additive model, even though they have an identical number of parameters, strongly suggest the importance of intramolecular polarization effects. In summary, this and previous works together show that the pGM models possess both accuracy and transferability, which are expected to serve as foundations for the development of next-generation polarizable force fields for modeling various polarization-sensitive biological systems and processes.

Accurate Reproduction of Quantum Mechanical Many-Body Interactions in Peptide Main-Chain Hydrogen-Bonding Oligomers by the Polarizable Gaussian Multipole Model.

A key advantage of polarizable force fields is their ability to model the atomic polarization effects that play key roles in the atomic many-body interactions. In this work, we assessed the accuracy of the recently developed polarizable Gaussian Multipole (pGM) models in reproducing quantum mechanical (QM) interaction energies, many-body interaction energies, as well as the nonadditive and additive contributions to the many-body interactions for peptide main-chain hydrogen-bonding conformers, using glycine dipeptide oligomers as the model systems. Two types of pGM models were considered, including that with (pGM-perm) and without (pGM-ind) permanent atomic dipoles. The performances of the pGM models were compared with several widely used force fields, including two polarizable (Amoeba13 and ff12pol) and three additive (ff19SB, ff15ipq, and ff03) force fields. Encouragingly, the pGM models outperform all other force fields in terms of reproducing QM interaction energies, many-body interaction energies, as well as the nonadditive and additive contributions to the many-body interactions, as measured by the root-mean-square errors (RMSEs) and mean absolute errors (MAEs). Furthermore, we tested the robustness of the pGM models against polarizability parameterization errors by employing alternative polarizabilities that are either scaled or obtained from other force fields. The results show that the pGM models with alternative polarizabilities exhibit improved accuracy in reproducing QM many-body interaction energies as well as the nonadditive and additive contributions compared with other polarizable force fields, suggesting that the pGM models are robust against the errors in polarizability parameterizations. This work shows that the pGM models are capable of accurately modeling polarization effects and have the potential to serve as templates for developing next-generation polarizable force fields for modeling various biological systems.

Neglected N-Truncated Amyloid-ß Peptide and Its Mixed Cu-Zn Complexes.

Amyloid-ß (Aß) peptides are involved in Alzheimer's disease (AD) development. The interactions of these peptides with copper and zinc ions also seem to be crucial for this pathology. Although Cu(II) and Zn(II) ions binding by Aß peptides has been scrupulously investigated, surprisingly, this phenomenon has not been so thoroughly elucidated for N-truncated Aß4-x-probably the most common version of this biomolecule. This negligence also applies to mixed Cu-Zn complexes. From the structural in silico analysis presented in this work, it appears that there are two possible mixed Cu-Zn(Aß4-x) complexes with different stoichiometries and, consequently, distinct properties. The Cu-Zn(Aß4-x) complex with 1:1:1 stoichiometry may have a neuroprotective superoxide dismutase-like activity. On the other hand, another mixed 2:1:2 Cu-Zn(Aß4-x) complex is perhaps a seed for toxic oligomers. Hence, this work proposes a novel research direction for our better understanding of AD development.

PyRESP: A Program for Electrostatic Parameterizations of Additive and Induced Dipole Polarizable Force Fields.

Molecular modeling at the atomic level has been applied in a wide range of biological systems. The widely adopted additive force fields typically use fixed atom-centered partial charges to model electrostatic interactions. However, the additive force fields cannot accurately model polarization effects, leading to unrealistic simulations in polarization-sensitive processes. Numerous efforts have been invested in developing induced dipole-based polarizable force fields. Whether additive atomic charge models or polarizable induced dipole models are used, proper parameterization of the electrostatic term plays a key role in the force field developments. In this work, we present a Python program called PyRESP for performing atomic multipole parameterizations by reproducing ab initio electrostatic potential (ESP) around molecules. PyRESP provides parameterization schemes for several electrostatic models, including the RESP model with atomic charges for the additive force fields and the RESP-ind and RESP-perm models with additional induced and permanent dipole moments for the polarizable force fields. PyRESP is a flexible and user-friendly program that can accommodate various needs during force field parameterizations for molecular modeling of any organic molecules.

Stress tensor and constant pressure simulation for polarizable Gaussian multipole model.

Our previous article has established the theory of molecular dynamics (MD) simulations for systems modeled with the polarizable Gaussian multipole (pGM) electrostatics [Wei et al., J. Chem. Phys. 153(11), 114116 (2020)]. Specifically, we proposed the covalent basis vector framework to define the permanent multipoles and derived closed-form energy and force expressions to facilitate an efficient implementation of pGM electrostatics. In this study, we move forward to derive the pGM internal stress tensor for constant pressure MD simulations with the pGM electrostatics. Three different formulations are presented for the flexible, rigid, and short-range screened systems, respectively. The analytical formulations were implemented in the SANDER program in the Amber package and were first validated with the finite-difference method for two different boxes of pGM water molecules. This is followed by a constant temperature and constant pressure MD simulation for a box of 512 pGM water molecules. Our results show that the simulation system stabilized at a physically reasonable state and maintained the balance with the externally applied pressure. In addition, several fundamental differences were observed between the pGM and classic point charge models in terms of the simulation behaviors, indicating more extensive parameterization is necessary to utilize the pGM electrostatics.

S-Nitrosylation of p62 Inhibits Autophagic Flux to Promote α-Synuclein Secretion and Spread in Parkinson's Disease and Lewy Body Dementia.

Dysregulation of autophagic pathways leads to accumulation of abnormal proteins and damaged organelles in many neurodegenerative disorders, including Parkinson's disease (PD) and Lewy body dementia (LBD). Autophagy-related dysfunction may also trigger secretion and spread of misfolded proteins, such as α-synuclein (α-syn), the major misfolded protein found in PD/LBD. However, the mechanism underlying these phenomena remains largely unknown. Here, we used cell-based models, including human induced pluripotent stem cell-derived neurons, CRISPR/Cas9 technology, and male transgenic PD/LBD mice, plus vetting in human postmortem brains (both male and female). We provide mechanistic insight into this pathologic pathway. We find that aberrant S-nitrosylation of the autophagic adaptor protein p62 causes inhibition of autophagic flux and intracellular buildup of misfolded proteins, with consequent secretion resulting in cell-to-cell spread. Thus, our data show that pathologic protein S-nitrosylation of p62 represents a critical factor not only for autophagic inhibition and demise of individual neurons, but also for α-syn release and spread of disease throughout the nervous system.SIGNIFICANCE STATEMENT In Parkinson's disease and Lewy body dementia, dysfunctional autophagy contributes to accumulation and spread of aggregated α-synuclein. Here, we provide evidence that protein S-nitrosylation of p62 inhibits autophagic flux, contributing to α-synuclein aggregation and spread.

Toxoplasma gondii serine hydrolases regulate parasite lipid mobilization during growth and replication within the host.

The intracellular protozoan parasite Toxoplasma gondii must scavenge cholesterol and other lipids from the host to facilitate intracellular growth and replication. Enzymes responsible for neutral lipid synthesis have been identified but there is no evidence for enzymes that catalyze lipolysis of cholesterol esters and esterified lipids. Here, we characterize several T. gondii serine hydrolases with esterase and thioesterase activities that were previously thought to be depalmitoylating enzymes. We find they do not cleave palmitoyl thiol esters but rather hydrolyze short-chain lipid esters. Deletion of one of the hydrolases results in alterations in levels of multiple lipids species. We also identify small-molecule inhibitors of these hydrolases and show that treatment of parasites results in phenotypic defects reminiscent of parasites exposed to excess cholesterol or oleic acid. Together, these data characterize enzymes necessary for processing lipids critical for infection and highlight the potential for targeting parasite hydrolases for therapeutic applications.

Determining the atomic charge of calcium ion requires the information of its coordination geometry in an EF-hand motif.

It is challenging to parameterize the force field for calcium ions (Ca2+) in calcium-binding proteins because of their unique coordination chemistry that involves the surrounding atoms required for stability. In this work, we observed a wide variation in Ca2+ binding loop conformations of the Ca2+-binding protein calmodulin, which adopts the most populated ternary structures determined from the molecular dynamics simulations, followed by ab initio quantum mechanical (QM) calculations on all 12 amino acids in the loop that coordinate Ca2+ in aqueous solution. Ca2+ charges were derived by fitting to the electrostatic potential in the context of a classical or polarizable force field (PFF). We discovered that the atomic radius of Ca2+ in conventional force fields is too large for the QM calculation to capture the variation in the coordination geometry of Ca2+ in its ionic form, leading to unphysical charges. Specifically, we found that the fitted atomic charges of Ca2+ in the context of PFF depend on the coordinating geometry of electronegative atoms from the amino acids in the loop. Although nearby water molecules do not influence the atomic charge of Ca2+, they are crucial for compensating for the coordination of Ca2+ due to the conformational flexibility in the EF-hand loop. Our method advances the development of force fields for metal ions and protein binding sites in dynamic environments.

S-nitrosylated TDP-43 triggers aggregation, cell-to-cell spread, and neurotoxicity in hiPSCs and in vivo models of ALS/FTD.

Rare genetic mutations result in aggregation and spreading of cognate proteins in neurodegenerative disorders; however, in the absence of mutation (i.e., in the vast majority of "sporadic" cases), mechanisms for protein misfolding/aggregation remain largely unknown. Here, we show environmentally induced nitrosative stress triggers protein aggregation and cell-to-cell spread. In patient brains with amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), aggregation of the RNA-binding protein TDP-43 constitutes a major component of aberrant cytoplasmic inclusions. We identify a pathological signaling cascade whereby reactive nitrogen species cause S-nitrosylation of TDP-43 (forming SNO-TDP-43) to facilitate disulfide linkage and consequent TDP-43 aggregation. Similar pathological SNO-TDP-43 levels occur in postmortem human FTD/ALS brains and in cell-based models, including human-induced pluripotent stem cell (hiPSC)-derived neurons. Aggregated TDP-43 triggers additional nitrosative stress, representing positive feed forward leading to further SNO-TDP-43 formation and disulfide-linked oligomerization/aggregation. Critically, we show that these redox reactions facilitate cell spreading in vivo and interfere with the TDP-43 RNA-binding activity, affecting SNMT1 and phospho-(p)CREB levels, thus contributing to neuronal damage in ALS/FTD disorders.

Quantitative profiling of protease specificity.

Proteases are an important class of enzymes, whose activity is central to many physiologic and pathologic processes. Detailed knowledge of protease specificity is key to understanding their function. Although many methods have been developed to profile specificities of proteases, few have the diversity and quantitative grasp necessary to fully define specificity of a protease, both in terms of substrate numbers and their catalytic efficiencies. We have developed a concept of "selectome"; the set of substrate amino acid sequences that uniquely represent the specificity of a protease. We applied it to two closely related members of the Matrixin family-MMP-2 and MMP-9 by using substrate phage display coupled with Next Generation Sequencing and information theory-based data analysis. We have also derived a quantitative measure of substrate specificity, which accounts for both the number of substrates and their relative catalytic efficiencies. Using these advances greatly facilitates elucidation of substrate selectivity between closely related members of a protease family. The study also provides insight into the degree to which the catalytic cleft defines substrate recognition, thus providing basis for overcoming two of the major challenges in the field of proteolysis: 1) development of highly selective activity probes for studying proteases with overlapping specificities, and 2) distinguishing targeted proteolysis from bystander proteolytic events.

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with endemic human coronaviruses.

The SARS-CoV-2 proteome shares regions of conservation with endemic human coronaviruses (CoVs), but it remains unknown to what extent these may be cross-recognized by the antibody response. Here, we study cross-reactivity using a highly multiplexed peptide assay (PepSeq) to generate an epitope-resolved view of IgG reactivity across all human CoVs in both COVID-19 convalescent and negative donors. PepSeq resolves epitopes across the SARS-CoV-2 Spike and Nucleocapsid proteins that are commonly targeted in convalescent donors, including several sites also recognized in some uninfected controls. By comparing patterns of homologous reactivity between CoVs and using targeted antibody-depletion experiments, we demonstrate that SARS-CoV-2 elicits antibodies that cross-recognize pandemic and endemic CoV antigens at two Spike S2 subunit epitopes. We further show that these cross-reactive antibodies preferentially bind endemic homologs. Our findings highlight sites at which the SARS-CoV-2 response appears to be shaped by previous CoV exposures and which have the potential to raise broadly neutralizing responses.

Efficient formulation of polarizable Gaussian multipole electrostatics for biomolecular simulations.

Molecular dynamics simulations of biomolecules have been widely adopted in biomedical studies. As classical point-charge models continue to be used in routine biomolecular applications, there have been growing demands on developing polarizable force fields for handling more complicated biomolecular processes. Here, we focus on a recently proposed polarizable Gaussian Multipole (pGM) model for biomolecular simulations. A key benefit of pGM is its screening of all short-range electrostatic interactions in a physically consistent manner, which is critical for stable charge-fitting and is needed to reproduce molecular anisotropy. Another advantage of pGM is that each atom's multipoles are represented by a single Gaussian function or its derivatives, allowing for more efficient electrostatics than other Gaussian-based models. In this study, we present an efficient formulation for the pGM model defined with respect to a local frame formed with a set of covalent basis vectors. The covalent basis vectors are chosen to be along each atom's covalent bonding directions. The new local frame can better accommodate the fact that permanent dipoles are primarily aligned along covalent bonds due to the differences in electronegativity of bonded atoms. It also allows molecular flexibility during molecular simulations and facilitates an efficient formulation of analytical electrostatic forces without explicit torque computation. Subsequent numerical tests show that analytical atomic forces agree excellently with numerical finite-difference forces for the tested system. Finally, the new pGM electrostatics algorithm is interfaced with the particle mesh Ewald (PME) implementation in Amber for molecular simulations under the periodic boundary conditions. To validate the overall pGM/PME electrostatics, we conducted an NVE simulation for a small water box of 512 water molecules. Our results show that to achieve energy conservation in the polarizable model, it is important to ensure enough accuracy on both PME and induction iteration. It is hoped that the reformulated pGM model will facilitate the development of future force fields based on the pGM electrostatics for applications in biomolecular systems and processes where polarization plays crucial roles.

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with an endemic human CoV.

A high-resolution understanding of the antibody response to SARS-CoV-2 is important for the design of effective diagnostics, vaccines and therapeutics. However, SARS-CoV-2 antibody epitopes remain largely uncharacterized, and it is unknown whether and how the response may cross-react with related viruses. Here, we use a multiplexed peptide assay ('PepSeq') to generate an epitope-resolved view of reactivity across all human coronaviruses. PepSeq accurately detects SARS-CoV-2 exposure and resolves epitopes across the Spike and Nucleocapsid proteins. Two of these represent recurrent reactivities to conserved, functionally-important sites in the Spike S2 subunit, regions that we show are also targeted for the endemic coronaviruses in pre-pandemic controls. At one of these sites, we demonstrate that the SARS-CoV-2 response strongly and recurrently cross-reacts with the endemic virus hCoV-OC43. Our analyses reveal new diagnostic and therapeutic targets, including a site at which SARS-CoV-2 may recruit common pre-existing antibodies and with the potential for broadly-neutralizing responses.

The Antimalarial Natural Product Salinipostin A Identifies Essential α/ß Serine Hydrolases Involved in Lipid Metabolism in P. falciparum Parasites.

Salinipostin A (Sal A) is a potent antiplasmodial marine natural product with an undefined mechanism of action. Using a Sal A-derived activity-based probe, we identify its targets in the Plasmodium falciparum parasite. All of the identified proteins contain α/ß serine hydrolase domains and several are essential for parasite growth. One of the essential targets displays a high degree of homology to human monoacylglycerol lipase (MAGL) and is able to process lipid esters including a MAGL acylglyceride substrate. This Sal A target is inhibited by the anti-obesity drug Orlistat, which disrupts lipid metabolism. Resistance selections yielded parasites that showed only minor reductions in sensitivity and that acquired mutations in a PRELI domain-containing protein linked to drug resistance in Toxoplasma gondii. This inability to evolve efficient resistance mechanisms combined with the non-essentiality of human homologs makes the serine hydrolases identified here promising antimalarial targets.

Interactions between motor domains in kinesin-14 Ncd - a molecular dynamics study.

Minus-end directed, non-processive kinesin-14 Ncd is a dimeric protein with C-terminally located motor domains (heads). Generation of the power-stroke by Ncd consists of a lever-like rotation of a long superhelical 'stalk' segment while one of the kinesin's heads is bound to the microtubule. The last ∼30 amino acids of Ncd head play a crucial but still poorly understood role in this process. Here, we used accelerated molecular dynamics simulations to explore the conformational dynamics of several systems built upon two crystal structures of Ncd, the asymmetrical T436S mutant in pre-stroke/post-stroke conformations of two partner subunits and the symmetrical wild-type protein in pre-stroke conformation of both subunits. The results revealed a new conformational state forming following the inward motion of the subunits and stabilized with several hydrogen bonds to residues located on the border or within the C-terminal linker, i.e. a modeled extension of the C-terminus by residues 675-683. Forming of this new, compact Ncd conformation critically depends on the length of the C-terminus extending to at least residue 681. Moreover, the associative motion leading to the compact conformation is accompanied by a partial lateral rotation of the stalk. We propose that the stable compact conformation of Ncd may represent an initial state of the working stroke.