Yasuaki Komuro

Dimensionality of Collective Variables for Describing Conformational Changes of a Multi-Domain Protein.

Collective variables (CVs) are often used in molecular dynamics simulations based on enhanced sampling algorithms to investigate large conformational changes of a protein. The choice of CVs in these simulations is essential because it affects simulation results and impacts the free-energy profile, the minimum free-energy pathway (MFEP), and the transition-state structure. Here we examine how many CVs are required to capture the correct transition-state structure during the open-to-close motion of adenylate kinase using a coarse-grained model in the mean forces string method to search the MFEP. Various numbers of large amplitude principal components are tested as CVs in the simulations. The incorporation of local coordinates into CVs, which is possible in higher dimensional CV spaces, is important for capturing a reliable MFEP. The Bayesian measure proposed by Best and Hummer is sensitive to the choice of CVs, showing sharp peaks when the transition-state structure is captured. We thus evaluate the required number of CVs needed in enhanced sampling simulations for describing protein conformational changes.

Rational design of crystal contact-free space in protein crystals for analyzing spatial distribution of motions within protein molecules.

Contacts with neighboring molecules in protein crystals inevitably restrict the internal motions of intrinsically flexible proteins. The resultant clear electron densities permit model building, as crystallographic snapshot structures. Although these still images are informative, they could provide biased pictures of the protein motions. If the mobile parts are located at a site lacking direct contacts in rationally designed crystals, then the amplitude of the movements can be experimentally analyzed. We propose a fusion protein method, to create crystal contact‐free space (CCFS) in protein crystals and to place the mobile parts in the CCFS. Conventional model building fails when large amplitude motions exist. In this study, the mobile parts appear as smeared electron densities in the CCFS, by suitable processing of the X‐ray diffraction data. We applied the CCFS method to a highly mobile presequence peptide bound to the mitochondrial import receptor, Tom20, and a catalytically relevant flexible segment in the oligosaccharyltransferase, AglB. These two examples demonstrated the general applicability of the CCFS method to the analysis of the spatial distribution of motions within protein molecules.

CHARMM Force-Fields with Modified Polyphosphate Parameters Allow Stable Simulation of the ATP-Bound Structure of Ca(2+)-ATPase.

Adenosine triphosphate (ATP) is an indispensable energy source in cells. In a wide variety of biological phenomena like glycolysis, muscle contraction/relaxation, and active ion transport, chemical energy released from ATP hydrolysis is converted to mechanical forces to bring about large-scale conformational changes in proteins. Investigation of structure-function relationships in these proteins by molecular dynamics (MD) simulations requires modeling of ATP in solution and ATP bound to proteins with accurate force-field parameters. In this study, we derived new force-field parameters for the triphosphate moiety of ATP based on the high-precision quantum calculations of methyl triphosphate. We tested our new parameters on membrane-embedded sarcoplasmic reticulum Ca(2+)-ATPase and four soluble proteins. The ATP-bound structure of Ca(2+)-ATPase remains stable during MD simulations, contrary to the outcome in shorter simulations using original parameters. Similar results were obtained with the four ATP-bound soluble proteins. The new force-field parameters were also tested by investigating the range of conformations sampled during replica-exchange MD simulations of ATP in explicit water. Modified parameters allowed a much wider range of conformational sampling compared with the bias toward extended forms with original parameters. A diverse range of structures agrees with the broad distribution of ATP conformations in proteins deposited in the Protein Data Bank. These simulations suggest that the modified parameters will be useful in studies of ATP in solution and of the many ATP-utilizing proteins.

Intentional crystal-contact-free space in protein crystal

To understand the function of proteins, it is essential to perform the structural analysis of the protein complexes with ligands, such as substrates or partner molecules. The motions of ligands are restricted by the contacts with neighbor protein molecules in the crystal lattice. Here, we propose a new technique to analyze dynamics of a ligand in the bound state preserved in the crystal-contact-free space, which is intentionally created in protein crystals. We used Tom20 as a target protein. Tom20 functions as a general protein import receptor, by recognizing N-terminal signal sequences (presequences) of mitochondrial matrix proteins. Our working hypothesis is that the promiscuous specificity of Tom20 is attributed to the large mobility of the presequneces in the binding groove of Tom20 (1,2). Our aim is to obtain electron density that reflects the large mobility of a presequence in the crystal-contact-free space. In order to create the crystal-contact-free space, we took advantage of a protein fused with maltose binding protein (MBP). The key of the design is the connection of the two proteins firmly. We fused the C-terminal α-helix of MBP and the N-terminal α-helix of Tom20 seamlessly. After a systematic model building study, we decided to use a design with four residues inserted in the linker region. We found smeared electron density in the binding site of presequences in the difference Fourier electron-density map. We attached an iodine atom at the N-terminus of the presequence and confirmed the N-terminal position in the smeared electron density. We performed molecular dynamics simulation without the tethering in solution (3). The electron density simulated from the MD trajectory was fully consistent with the smeared electron density in the crystal contact-free space. We concluded that the smeared electron density corresponded to the partially overlapping region of the multiple states of the bound presequence.