15 Search Results

Permselective nanochannels are ubiquitous in biological systems, controlling ion transport and maintaining a potential difference across a cell surface. Surface layers (S-layers) are proteinaceous, generally charged lattices punctuated with nanoscale pores that form the outermost cell envelope component of virtually all archaea and many bacteria. Ammonia oxidizing archaea (AOA) obtain their energy exclusively from oxidizing ammonia directly below the S-layer lattice, but how the charged surfaces and nanochannels affect availability of NH4+ at the reaction site is unknown. In this report, we examine the electrochemical properties of negatively charged S-layers for asymmetrically forced ion transport governed by Michaelis–Menten kinetics atmore » ultralow concentrations. Our 3-dimensional electrodiffusion reaction simulations revealed that a negatively charged S-layer can invert the potential across the nanochannel to favor chemically forced NH₄⁺ transport, analogous to polarity switching in nanofluidic field-effect transistors. Polarity switching was not observed when only the interior of the nanochannels was charged. We found that S-layer charge, nanochannel geometry, and enzymatic turnover rate are finely tuned to elevate NH₄⁺ concentration at the active site, potentially enabling AOA to occupy nutrient-poor ecological niches. Strikingly, and in contrast to voltage-biased systems, magnitudes of the co- and counterion currents in the charged nanochannels were nearly equal and amplified disproportionally to the NH₄⁺ current. These simulations imply that engineered arrays of crystalline proteinaceous membranes could find unique applications in industrial energy conversion or separation processes.« less

We report that cysteine is a rare but functionally important amino acid that is often subject to covalent modification. Cysteine oxidation plays an important role in many human disease processes, and basal levels of cysteine oxidation are required for proper cellular function. Because reactive cysteine residues are typically ionized to the thiolate anion (Cys-S^-), their formation of a covalent bond alters the electrostatic and steric environment of the active site. X-ray-induced photo-oxidation to sulfenic acids (Cys-SOH) can recapitulate some aspects of the changes that occur under physiological conditions. Here we propose how site-specific cysteine photo-oxidation can be used to interrogatemore » ensuing changes in protein structure and dynamics at atomic resolution. Although this powerful approach can connect cysteine covalent modification to global protein conformational changes and function, careful biochemical validation must accompany all such studies to exclude misleading artifacts. Lastly, new types of X-ray crystallography experiments and powerful computational methods are creating new opportunities to connect conformational dynamics to catalysis for the large class of systems that use covalently modified cysteine residues for catalysis or regulation.« less

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, andmore » they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.« less

Elastic network models (ENMs) and constraint-based, topological rigidity analysis are two distinct, coarse-grained approaches to study conformational flexibility of macromolecules. In the two decades since their introduction, both have contributed significantly to insights into protein molecular mechanisms and function. However, despite a shared purpose of these approaches, the topological nature of rigidity analysis, and thereby the absence of motion modes, has impeded a direct comparison. We present an alternative, kinematic approach to rigidity analysis, which circumvents these drawbacks. We introduce a novel protein hydrogen bond network spectral decomposition, which provides an orthonormal basis for collective motions modulated by noncovalent interactions,more » analogous to the eigenspectrum of normal modes. The zero modes decompose proteins into rigid clusters identical to those from topological rigidity, while nonzero modes rank protein motions by their hydrogen bond collective energy penalty. Our kinematic flexibility analysis bridges topological rigidity theory and ENM, enabling a detailed analysis of motion modes obtained from both approaches. Analysis of a large, structurally diverse data set revealed that collectivity of protein motions, reported by the Shannon entropy, is significantly reduced for rigidity theory compared to normal mode approaches. Strikingly, kinematic flexibility analysis suggests that the hydrogen bonding network encodes a protein-fold specific, spatial hierarchy of motions, which goes nearly undetected in ENM. This hierarchy reveals distinct motion regimes that rationalize experimental and simulated protein stiffness variations. Kinematic motion modes highly correlate with reported crystallographic B factors and molecular dynamics simulations of adenylate kinase. A formal expression for changes in free energy derived from the spectral decomposition indicates that motions across nearly 40% of modes obey enthalpy–entropy compensation. Taken together, our results suggest that hydrogen bond networks have evolved to modulate protein structure and dynamics, which can be efficiently probed by kinematic flexibility analysis.« less

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. We report, ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteinemore » thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.« less

Helicobacter pylori infects nearly half of the world’s population and is the primary cause of various gastric diseases. It has evolved various virulence factors to aid its host colonization and infection, including the vacuolating cytotoxin A (VacA) that is responsible for the pathogenesis of H. pylori-related diseases. Here, we resolve multiple structures of the water-soluble VacA oligomeric assemblies using cryoelectron microscopy (cryo-EM) at near-atomic resolution. These studies suggest a model of structural changes of functional VacA hexamer needed for the pore-formation process across the membrane and highlight the capability of cryo-EM to resolve multiple structure snapshots from a single specimenmore » at near-atomic resolution.« less

Here, proteins and ligands sample a conformational ensemble that governs molecular recognition, activity, and dissociation. In structure-based drug design, access to this conformational ensemble is critical to understand the balance between entropy and enthalpy in lead optimization. However, ligand conformational heterogeneity is currently severely underreported in crystal structures in the Protein Data Bank, owing in part to a lack of automated and unbiased procedures to model an ensemble of protein–ligand states into X-ray data. Here, we designed a computational method, qFit-ligand, to automatically resolve conformationally averaged ligand heterogeneity in crystal structures, and applied it to a large set of proteinmore » receptor–ligand complexes. In an analysis of the cancer related BRD4 domain, we found that up to 29% of protein crystal structures bound with drug-like molecules present evidence of unmodeled, averaged, relatively isoenergetic conformations in ligand–receptor interactions. In many retrospective cases, these alternate conformations were adventitiously exploited to guide compound design, resulting in improved potency or selectivity. Combining qFit-ligand with high-throughput screening or multitemperature crystallography could therefore augment the structure-based drug design toolbox.« less

The function of protein, RNA, and DNA is modulated by fast, dynamic exchanges between three-dimensional conformations. Conformational sampling of biomolecules with exact and nullspace inverse kinematics, using rotatable bonds as revolute joints and noncovalent interactions as holonomic constraints, can accurately characterize these native ensembles. However, sampling biomolecules remains challenging owing to their ultra-high dimensional configuration spaces, and the requirement to avoid (self-) collisions, which results in low acceptance rates. In this paper, we present two novel mechanisms to overcome these limitations. First, we introduce temporary constraints between near-colliding links. The resulting constraint varieties instantaneously redirect the search for collision-free conformations,more » and couple motions between distant parts of the linkage. Second, we adapt a randomized Poisson-disk motion planner, which prevents local oversampling and widens the search, to ultra-high dimensions. Tests on several model systems show that the sampling acceptance rate can increase from 16% to 70%, and that the conformational coverage in loop modeling measured as average closeness to existing loop conformations doubled. Finally, correlated protein motions identified with our algorithm agree with those from MD simulations.« less

Proteins exist as conformational ensembles, exchanging between substates to perform their function. Advances in experimental techniques yield unprecedented access to structural snapshots of their conformational landscape. However, computationally modeling how proteins use collective motions to transition between substates is challenging owing to a rugged landscape and large energy barriers. Here in this paper, we present a new, robotics-inspired motion planning procedure called dCCRRT that navigates the rugged landscape between substates by introducing dynamic, interatomic constraints to modulate frustration. The constraints balance non-native contacts and flexibility, and instantaneously redirect the motion towards sterically favorable conformations. On a test set of eightmore » proteins determined in two conformations separated by, on average, 7.5Å root mean square deviation (RMSD), our pathways reduced the Cα atom RMSD to the goal conformation by 78%, outperforming peer methods. Additionally, we then applied dCC-RRT to examine how collective, small-scale motions of four side-chains in the active site of cyclophilin A propagate through the protein. dCC-RRT uncovered a spatially contiguous network of residues linked by steric interactions and collective motion connecting the active site to a recently proposed, non-canonical capsid binding site 25Å away, rationalizing NMR and multi-temperature crystallography experiments. In all, dCC-RRT can reveal detailed, all-atom molecular mechanisms for small and large amplitude motions.Source code and binaries are freely available at https://github.com/ExcitedStates/KGS/.« less

Surface layers (S-layers) are two-dimensional, proteinaceous, porous lattices that form the outermost cell envelope component of virtually all archaea and many bacteria. Despite exceptional sequence diversity, S-layer proteins (SLPs) share important characteristics such as their ability to form crystalline sheets punctuated with nano-scale pores, and their propensity for charged amino acids, leading to acidic or basic isoelectric points. However, the precise function of S-layers, or the role of charged SLPs and how they relate to cellular metabolism is unknown. Nano-scale lattices affect the diffusion behavior of low-concentration solutes, even if they are significantly smaller than the pore size. Here, wemore » offer a rationale for charged S-layer proteins in the context of the structural evolution of S-layers. Using the ammonia-oxidizing archaea (AOA) as a model for S-layer geometry, and a 2D electrodiffusion reaction computational framework to simulate diffusion and consumption of the charged solute ammonium (NH₄⁺), we find that the characteristic length scales of nanoporous S-layers elevate the concentration of NH₄⁺ in the pseudo-periplasmic space. Our simulations suggest an evolutionary, mechanistic basis for S-layer charge and shed light on the unique ability of some AOA to oxidize ammonia in environments with nanomolar NH₄⁺ availability, with broad implications for comparisons of ecologically distinct populations.« less