Finding Gas Diffusion Pathways in Proteins: Application to O2 and H2 Transport in CpI [FeFe]-Hydrogenase and the Role of Packing Defects

Complementary Structural Mass Spectrometry Techniques Reveal Local Dynamics in Functionally Important Regions of a Metastable Serpin

Ligand Migration and Cavities within Scapharca Dimeric HbI: Studies by Time-Resolved Crystallo- graphy, Xe Binding, and Computational Analysis

Recent development in static and dynamic structure analyses of polymer crystals has been reviewed. Various methods were developed to enhance the reliability of static structure analysis. Usage of synchrotron high-energy X-ray beam allowed us to increase the total number of observed X-ray reflections by one order. Wide-angle neutron diffraction revealed the hydrogen atomic positions accurately, making it possible to evaluate the mechanical property of polymer crystals quantitatively. Time-resolved measurements of wide-angle and small-angle X-ray scatterings as well as infrared and Raman spectra have revealed the structural revolution processes as seen in the studies of isothermal crystallization and mechanical deformation processes.

Atomistic molecular dynamics simulations of human serum albumin in the presence and absence of disulfide bonds are presented. Simulations of 70 ns duration provide information on the relevance of disulfide bonds in the dynamics and structural conformation of HSA. Significant conformational changes are observed in the absence of disulfide bonds after 35 ns that could impact the functionality and stability of the protein. Changes in the secondary structure, hydrogen bonds, B factors, and cross-correlations reveal which disulfide bonds are important for keeping the secondary and tertiary structure and dynamics of the protein (e.g., Cys168-Cys177, Cys278-Cys289) and which have little effect on the local structure and dynamics (e.g., Cys200-Cys246, Cys461-Cys477). Removing all disulfide bonds in the protein appears to be a practical prescreening tool for identifying disulfide bonds relevant to structure and dynamics. In the absence of disulfide bonds, certain hydrogen bonds and correlated motions vanish, affecting the structure of neighboring residues. The structure of the primary binding sites of HSA is partially affected when disulfide bonds are removed. For the native structure, simulations clearly reveal the conformational changes that allow the only free cysteine to be exposed on the protein surface to form intermolecular disulfide bonds; this information could not be resolved from the static crystal structure alone. The absence of specific disulfide bonds could lead to partially unfolded structures; such structures are known to be prone to protein aggregation. Removing disulfide bonds could have similar consequences in other proteins of interest, such as immunoglobulin G.

Ebola viruses (EBOV) will induce acute hemorrhagic fever, which is fatal to humans and nonhuman primates. The combination of EBOV VP35 peptide with nucleoprotein N-terminal (NPNTD) is proposed based on static crystal structures in recent studies, but VP35 binding mechanism and conformational dynamics are still unclear. This investigation, using Molecular Dynamic (MD) simulation and Molecular Mechanics Generalized Born Surface Area (MM-GB/SA) energy calculation, more convincingly proves the greater roles of the protein binding mechanisms than do hints from the static crystal structure observations. Conformational analysis of the systems demonstrate that combination with VP35 may lead to the conformational transition of NPNTD from “open” to “closed” state. According to the analyses of binding free energies and their decomposition, VP35 residue R37 plays a crucial role in wild type as well as mutant systems. Mutations of I29 and L33 to aspartate as well as M34 to proline affect binding affinity mainly through influencing electrostatic interaction, which is closely related to H-bonds formation. In addition, mutations mainly affect β-hairpin and loop regions, among which, M34P may have the greatest influence to the binding. This study may provide specific binding mechanisms between VP35 peptide and NPNTD, especially some important residues concerning binding.

Insights into How Mutations Thermostabilize G-Protein-Coupled Receptors

The equilibrium angles and distributions of chi(1) rotamers for mobile surface side chains of the small, 63-residue, B1 domain of protein L have been calculated from the static crystal structure by rigid body/torsion angle simulated annealing using a torsion angle database potential of mean force and compared to those deduced by Monte Carlo analysis of side chain residual dipolar couplings measured in solution. Good agreement between theory and experiment is observed, indicating that for side chains undergoing rotamer averaging that is fast on the chemical shift time scale, the equilibrium angles and distribution of chi(1) rotamers are largely determined by the backbone phi/psi torsion angles.

A topochemical polymerization governed by a topotactic polymorphic transition is reported. A monomer functionalized with azide and an internal alkyne crystallized as an unreactive polymorph with two molecules in the asymmetric unit. The molecules are aligned in a head-to-head fashion, thereby avoiding the azide-alkyne proximity for the topochemical azide-alkyne cycloaddition (TAAC) reaction. However, upon heating, one of the two conformers underwent a drastic 180° rotation, leading to a single-crystal-to-single-crystal (SCSC) polymorphic transition to a reactive form, wherein the molecules are head-to-tail arranged, ensuring azide-alkyne proximity. The new polymorph underwent TAAC reaction to form a trisubstituted 1,2,3-triazole-linked polymer. These results, showing unexpected topochemical reactivity of a crystal due to the intermediacy of an SCSC polymorphic transition from an unreactive form to a reactive form, highlight that predicting topochemical reactivity by relying on the static crystal structure can be misleading.

How Can Mutations Thermostabilize G-Protein-Coupled Receptors?

The intracellular domain of clusters of differentiation 44 (CD44) binding to the FERM (protein 4.1-ezrin-radixin-moesin) domain of ERM (ezrin/radixin/moesin) proteins and furthermore triggering the recruitment of spleen tyrosine kinase (Syk) are very important in the process of tumor cell adhesion, migration and proliferation. At first, it was found that CD44/FERM structure was stable by observing CD44/FERM complex conformation and analyzing the interaction of interface residues both in static crystal structure and in equilibrium process. Meanwhile, unconventional immunoreceptor tyrosine-based activation motif (ITAM-like), and phosphorylation sites Y191 and Y205 were buried in FERM domain, which would hinder the phosphorylation of ERM proteins, the recruitment of Syk and subsequent signal transduction. Then, steered molecular dynamics simulation was applied to simulate the interaction between CD44 and FERM domain in the mechanical environment. The results showed that mechanical signal could induce the exposure of the ITAM-like motif and phosphorylation site Y205 by tracking and analyzing CD44/FERM complex conformational changes and the solvent-accessible surface area. This study revealed how the force regulates the activation of downstream signal through CD44 intracellular domain for the first time, and would be useful for further understanding the adhesion and migration pathway of cancer cells and the design of antitumor drugs.

Mechanisms of electron decoherence at surfaces are manifold as they may originate from the various complex interactions of electrons with the static crystal structure and dynamical degrees of freedom of the environment. Decoherence effects manifest themselves in the spectroscopic data in a convoluted fashion and it is usually hard or even impossible to fully disentangle them from each other because of the lack of control of underlying mechanisms by the external observer. However, electronic propagation in quasi‐two‐dimensional image potential bands (IS‐bands) on flat low index surfaces of some metals is subject to an efficient decoherence mechanism that can be controlled externally by careful preparation of the surface. By dosing the concentration (i.e. the coverage) of adsorbates on clean surfaces, which act as randomly distributed scattering centres, one can tune the strength of incoherent IS‐electron scattering from defects. Such processes in IS‐bands on Cu(100) surface have been investigated by two‐photon‐photoemission (2PPE) spectroscopy and interpreted using Fermi golden rule approach to calculation of the quasiparticle decay rates and scattering cross sections. However, these results could reproduce the experimental data only in a limited energy interval. Here, we employ the description of electron decoherence in IS‐bands based on the propagator approach and infrared renormalization of quasiparticle self‐energy and demonstrate that it gives a very good agreement between the theoretical and experimental results for the cross sections. This enables us to discuss the temporal stages of electron dynamics and decoherence in the intermediate states of 2PPE spectroscopy of surface bands.

Avelumab, approved by the US Food and Drug Administration (FDA) for the treatment of Merkel cell carcinoma in adults and paediatric patients in 2017, is an investigational fully human anti–PD-L1 IgG1 antibody that inhibits PD-1/PD-L1 interactions. Although the crystal structure of the avelumab/PD-L1 complex was reported in 2017, which provided us the interface information at atom level, the dynamics information of the complex is missed, and some key residues could not be detected in that static crystal structure. Here, molecular dynamics simulations were performed for the avelumab/PD-L1 complex to map the epitope to paratope residues. The results showed that the epitope residues locating on the C strand (PD-L1TYR56 and PD-L1GLU58), CC’ loop (PD-L1GLU60, PD-L1ASP61 and PD-L1LYS62), C’ strand (PD-L1ASN63), and C'D loop (PD-L1HIS69) of PD-L1 mainly form the interface with avelumab. The paratope residues on avelumab include TYR52H, SER54H, GLY102H, THR105H, TYR34L, ASP52L and ARG99L. The C’ strand of PD-L1 is also a binding region for PD-1. Thus, antibody avelumab block PD-1/PD-L1 interaction through direct competitive binding of the C’ strand of PD-L1.

Conformational Dynamics of a Membrane Transport Protein Probed by H/D Exchange and Covalent Labeling: The Glycerol Facilitator

The Mg-dependent 5-epi-aristolochene synthase from Nicotiana tabacum (called TEAS) could catalyze the linear farnesyl pyrophosphate (FPP) substrate to form bicyclic hydrocarbon 5-epi-aristolochene. The cyclization reaction mechanism of TEAS was proposed based on static crystal structures and quantum chemistry calculations in a few previous studies, but substrate FPP binding kinetics and protein conformational dynamics responsible for the enzymatic catalysis are still unclear. Herein, by elaborative and extensive molecular dynamics simulations, the loop conformation change and several crucial residues promoting the cyclization reaction in TEAS are elucidated. It is found that the unusual noncatalytic NH2-terminal domain is essential to stabilize Helix-K and the adjoining J-K loop of the catalytic COOH-terminal domain. It is also illuminated that the induce-fit J-K/A-C loop dynamics is triggered by Y527 and the optimum substrate binding mode in a "U-shape" conformation. The U-shaped ligand binding pose is maintained well with the cooperative interaction of the three Mg(2+)-containing coordination shell and conserved residue W273. Furthermore, the conserved Arg residue pair R264/R266 and aromatic residue pair Y527/W273, whose spatial orientations are also crucial to promote the closure of the active site to a hydrophobic pocket, as well as to form π-stacking interactions with the ligand, would facilitate the carbocation migration and electrophilic attack involving the catalytic reaction. Our investigation more convincingly proves the greater roles of the protein local conformational dynamics than do hints from the static crystal structure observations. Thus, these findings can act as a guide to new protein engineering strategies on diversifying the sesquiterpene products for drug discovery.

The urgent need for new treatments for the chronic lung disease idiopathic pulmonary fibrosis (IPF) motivates research into antagonists of the RGD binding integrin αvβ6, a protein linked to the initiation and progression of the disease. Molecular dynamics (MD) simulations of αvβ6 in complex with its natural ligand, pro-TGF-β1, show the persistence over time of a bidentate Arg-Asp ligand-receptor interaction and a metal chelate interaction between an aspartate on the ligand and an Mg2+ ion in the active site. This is typical of RGD binding ligands. Additional binding site interactions, which are not observed in the static crystal structure, are also identified. We investigate an RGD mimetic, which serves as a framework for a series of potential αvβ6 antagonists. The scaffold includes a derivative of the widely utilized 1,8-naphthyridine moiety, for which we present force field parameters, to enable MD and relative free energy perturbation (FEP) simulations. The MD simulations highlight the importance of hydrogen bonding and cation-π interactions. The FEP calculations predict relative binding affinities, within 1.5 kcal mol-1, on average, of experiments.