Atomic Resolution Structural Information Research Articles

Engineered antigen-binding fragments for enhanced crystallization of antibody:antigen complexes.

The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.

A molecular basis for tetramer destabilization and aggregation of transthyretin Ala97Ser.

Transthyretin (TTR)-related amyloidosis (ATTR) is a syndrome of diseases characterized by the extracellular deposition of fibrillar materials containing TTR variants. Ala97Ser (A97S) is the major mutation reported in Taiwanese ATTR patients. Here, we combine atomic resolution structural information together with the biochemical data to demonstrate that substitution of polar Ser for a small hydrophobic side chain of Ala at residue 97 of TTR largely influences the local packing density of the FG-loop, thus leading to the conformational instability of native tetramer, the increased monomeric species, and thus the enhanced amyloidogenicity of apo-A97S. Based on calorimetric studies, the tetramer destabilization of A97S can be substantially altered by interacting with native stabilizers via similarly energetic patterns compared to that of wild-type (WT) TTR; however, stabilizer binding partially rearranges the networks of hydrogen bonding in TTR variants while FG-loops of tetrameric A97S still remain relatively flexible. Moreover, TTR in complexed with holo-retinol binding protein 4 is slightly influenced by the structural and dynamic changes of FG-loop caused by A97S substitution with an approximately five-fold difference in binding affinity. Collectively, our findings suggest that the amyloidogenic A97S mutation destabilizes TTR by increasing the flexibility of the FG-loop in the monomer, thus modulating the rate of amyloid fibrillization.

Inferring Conformational State of Myosin Motor in an Atomic Force Microscopy Image via Flexible Fitting Molecular Simulations.

High-speed atomic force microscopy (HS-AFM) is a powerful technique to image the structural dynamics of biomolecules. We can obtain atomic-resolution structural information from the measured AFM image by superimposing a structural model on the image. We previously developed a flexible fitting molecular dynamics (MD) simulation method that allows for modest conformational changes when superimposed on an AFM image. In this study, for a molecular motor, myosin V (which changes its chemical state), we examined whether the conformationally distinct state in each HS-AFM image could be inferred via flexible fitting MD simulation. We first built models of myosin V bound to the actin filament in two conformational states, the “down-up” and “down-down” states. Then, for the previously obtained HS-AFM image of myosin bound to the actin filament, we performed flexible-fitting MD simulations using the two states. By comparing the fitting results, we inferred the conformational and chemical states from the AFM image.

Biophysical applications in structural and molecular biology.

The main objective of structural biology is to model proteins and other biological macromolecules and link the structural information to function and dynamics. The biological functions of protein molecules and nucleic acids are inherently dependent on their conformational dynamics. Imaging of individual molecules and their dynamic characteristics is an ample source of knowledge that brings new insights about mechanisms of action. The atomic-resolution structural information on most of the biomolecules has been solved by biophysical techniques; either by X-ray diffraction in single crystals or by nuclear magnetic resonance (NMR) spectroscopy in solution. Cryo-electron microscopy (cryo-EM) is emerging as a new tool foranalysis of a larger macromolecule that couldn't be solved by X-ray crystallography or NMR. Now a day's low-resolution Cryo-EM is used in combination with either X-ray crystallography or NMR. The present review intends to provide updated information on applications like X-ray crystallography, cryo-EM and NMR which can be used independently and/or together in solving structures of biological macromolecules for our full comprehension of their biological mechanisms.

The role of NMR in leveraging dynamics and entropy in drug design.

Summary:Nuclear Magnetic Resonance (NMR) spectroscopy has contributed to structure-based drug development (SBDD) in a unique way compared to the other biophysical methods. The potency of a ligand binding to a protein is dictated by the binding free energy, which is an intricate interplay between entropy and enthalpy. In addition to providing the atomic resolution structural information, NMR can help to identify protein-ligand interactions that potentially contribute to the enthalpic component of the free energy. NMR can also illuminate dynamic aspects of the interaction, which correspond to the entropic term of the free energy. The ability of NMR to access both terms in the free energy equation stems from the suite of experiments developed to shed light on various aspects that contribute to both entropy and enthalpy, deepening our understanding of the biological function of macromolecules and assisting to target them in physiological conditions. Here we provide a brief account of the contribution of NMR to SBDD, highlighting hallmark examples and discussing the challenges that demand further method development. In the era of integrated biology, the unique ability of NMR to directly ascertain structural and dynamical aspects of macromolecule and monitor changes in these properties upon engaging a ligand can be combined with computational and other structural and biophysical methods to provide a more complete picture of the energetics of drug engagement with the target. Such efforts can be used to engineer better drugs.

Structure of a Protein-RNA Complex by Solid-State NMR Spectroscopy.

Solid‐state NMR (ssNMR) is applicable to high molecular‐weight (MW) protein assemblies in a non‐amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein–RNA complexes (RNP) solely from ssNMR data. Instead of using low‐sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical‐shift‐perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein–RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods.

Structure of a Protein–RNA Complex by Solid‐State NMR Spectroscopy

AbstractSolid‐state NMR (ssNMR) is applicable to high molecular‐weight (MW) protein assemblies in a non‐amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein–RNA complexes (RNP) solely from ssNMR data. Instead of using low‐sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical‐shift‐perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein–RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods.

Energetics of Calmodulin Recognition of a Skeletal Muscle Ryanodine Receptor Site

In skeletal muscle, the calcium-binding protein calmodulin (CaM) modulates the opening and closing of the ryanodine receptor (RyR1), the main ion channel responsible for calcium release from the sarcoplasmic reticulum during excitation-contraction coupling. By weakly activating RyR1 at low (nM) calcium and inhibiting it at high (µM) calcium, CaM acts as a feedback regulator of calcium levels during muscle contraction. A short region on RyR1 (residues 3614-3640, RyR1p) serves as an anchor for CaM. We investigated the energetics of CaM-RyR1p recognition using Förster resonance energy transfer in auto-fluorescent biosensor constructs (YFP-RyRp-CFP). Using this system, we systematically explored the roles of individual RyR1 residues and each CaM domain at the interface, both in the presence and the absence of calcium. The interplay between the processes of calcium- and target-binding to CaM was probed by quantifying the energy of interaction between wild-type or mutated RyR1p sequences and CaM mutants having non-functional domain-specific Ca2+-binding sites. At low calcium, the interaction is weak (low µM), and mutation Trp3620Ala in the N-terminus of RyR1p (but not mutation Tyr3636Ala in the C-terminus) renders it non-specific. At high calcium, the interaction is at least three orders of magnitude stronger, and CaM C-domain binding to the N-terminal half of RyRp is the main mediator of recognition. A CaM C-domain Ca2+-binding mutant and/or Trp3620Ala RyR1p mutation significantly decrease the binding affinity, while a CaM N-domain Ca2+-binding mutant and/or Tyr3636Ala RyRp mutation have weaker effects. The primary role of CaM C-domain at the interface is maintained in the Tyr3636Ala RyRp complex, whereas Trp3620Ala mutation almost equalizes the energetic contributions of the two CaM domains. Future NMR studies of co-expressed CaM-RyR1p complexes will complement the thermodynamic data with atomic-resolution structural information.

Bioinformatic analysis of riboswitch structures uncovers variant classes with altered ligand specificity

Riboswitches are RNAs that form complex, folded structures that selectively bind small molecules or ions. As with certain groups of protein enzymes and receptors, some riboswitch classes have evolved to change their ligand specificity. We developed a procedure to systematically analyze known riboswitch classes to find additional variants that have altered their ligand specificity. This approach uses multiple-sequence alignments, atomic-resolution structural information, and riboswitch gene associations. Among the discoveries are unique variants of the guanine riboswitch class that most tightly bind the nucleoside 2'-deoxyguanosine. In addition, we identified variants of the glycine riboswitch class that no longer recognize this amino acid, additional members of a rare flavin mononucleotide (FMN) variant class, and also variants of c-di-GMP-I and -II riboswitches that might recognize different bacterial signaling molecules. These findings further reveal the diverse molecular sensing capabilities of RNA, which highlights the potential for discovering a large number of additional natural riboswitch classes.

Stabilized HIV-1 envelope glycoprotein trimers for vaccine use.

Purpose of reviewTo provide an update on the latest developments in the field of HIV-1 antibody-based soluble envelope glycoprotein (Env) trimer design for vaccine use.Recent findingsThe development of soluble native-like HIV-1 Env trimer immunogens has moved the field of antibody-based vaccine design forward dramatically over the past few years with refinement of various stabilizing approaches. However, despite this progress, significant challenges remain. Firstly, although trimers are relatively stable in solution, they nevertheless sample different conformational states, some of which may be less relevant to binding and induction of broadly neutralizing antibodies (bNAbs). Secondly, these trimers expose unwanted immunodominant surfaces that may distract the adaptive immune response from recognizing more immunorecessive but conserved neutralization-relevant surfaces on the trimer. The availability of atomic-resolution structural information has allowed guided design of mutations that have further stabilized trimers and allowed reduced exposure of unwanted epitopes. Moreover, chemical cross-linking approaches that do not require structural information have also contributed to trimer stabilization and selection of particular conformational forms. However, current knowledge suggests that strategies additional to trimer stabilization will be required to elicit bNAb, including targeting naïve B cell receptors with specific immunogens, and guiding B cell lineages toward recognizing conserved surfaces on Env with high affinity.SummaryThis review will give a perspective on these challenges, and summarize current approaches to overcoming them with the aim of developing immunogens to elicit bNAb responses in humans by active vaccination.

Solid-state NMR structures of integral membrane proteins.

Solid-state NMR is unique for its ability to obtain three-dimensional structures and to measure atomic-resolution structural and dynamic information for membrane proteins in native lipid bilayers. An increasing number and complexity of integral membrane protein structures have been determined by solid-state NMR using two main methods. Oriented sample solid-state NMR uses macroscopically aligned lipid bilayers to obtain orientational restraints that define secondary structure and global fold of embedded peptides and proteins and their orientation and topology in lipid bilayers. Magic angle spinning (MAS) solid-state NMR uses unoriented rapidly spinning samples to obtain distance and torsion angle restraints that define tertiary structure and helix packing arrangements. Details of all current protein structures are described, highlighting developments in experimental strategy and other technological advancements. Some structures originate from combining solid- and solution-state NMR information and some have used solid-state NMR to refine X-ray crystal structures. Solid-state NMR has also validated the structures of proteins determined in different membrane mimetics by solution-state NMR and X-ray crystallography and is therefore complementary to other structural biology techniques. By continuing efforts in identifying membrane protein targets and developing expression, isotope labelling and sample preparation strategies, probe technology, NMR experiments, calculation and modelling methods and combination with other techniques, it should be feasible to determine the structures of many more membrane proteins of biological and biomedical importance using solid-state NMR. This will provide three-dimensional structures and atomic-resolution structural information for characterising ligand and drug interactions, dynamics and molecular mechanisms of membrane proteins under physiological lipid bilayer conditions.

C3-P-12“Minispindle” as a tool for analysis of individual microtubule behavior in a mitotic spindle

The V-ATPase from Thermus thermophiles is a rotary enzyme, that mediates the coupling between ATP synthesis in the soluble part (V1) and proton translocation across membrane through the membrane-embedded part (Vo). Atomic resolution structural information of the soluble catalytic region has not been obtained yet, and little is known about the high-resolution structure of the Vo. Single particle analysis by cryo-electron microscopy has advanced to a technique for high-resolution structural analysis recently. Thus, now we are trying to get high-resolution structures of both V1 and VoV1 (intact enzyme) using a cryo-electron microscope (Titan Krios) equipped with a direct electron detector (Falcon II). We prepared grids for cryo-electron microscopy using Vitrobot (FEI) with the recombinant V1 and the Vo solubilized by DDM. We used thin carbon-backing for Vo and collected the images from both samples using EPU (FEI). For the efficient data collection by EPU, we used Quantifoil R2/4 made by Mo with 200 mesh. It is known that the particles on the images by cryo-electron microscopy are subjected to a beam-induced movement and so we applied motion collection before the image processing by Relion. We will show the resulted structures and discuss about possible improvements to the technique. Images of the VoV1 holoenzymes C3-P-12 doi:10.1093/jmicro/dfv315

High-resolution imaging of biotite using focal series exit wavefunction restoration and the graphene mechanical exfoliation method

AbstractWe have applied mechanical exfoliation for the preparation of ultra-thin samples of the phyllosilicate mineral biotite. We demonstrate that the 'scotch tape' approach, which was made famous as an early method for production of single-atom-thick graphene, can be used for production of sheet-silicate specimens that are sufficiently thin to allow high-resolution transmission electron microscope (HRTEM) imaging to be achieved successfully while also being free from the specimen preparation artefacts that are often caused by ion-beam milling techniques. Exfoliation of the biotite parallel to the (001) planes has produced layers as thin as two structural TOT units thick (∼2 nm). The minimal specimen thickness enabled not only HRTEM imaging but also the application of subsequent exit wavefunction restoration to reveal the pristine biotite lattice. Exit wavefunction restoration recovers the full complex electron wave from a focal series of HRTEM images, removing the effects of coherent lens aberrations. This combination of methods therefore produces images in which the observed features are readily interpreted to obtain atomic resolution structural information.

Solution Structure of Yeast Rpn9: INSIGHTS INTO PROTEASOME LID ASSEMBLY

The regulatory particle (RP) of the 26 S proteasome functions in preparing polyubiquitinated substrates for degradation. The lid complex of the RP contains an Rpn8-Rpn11 heterodimer surrounded by a horseshoe-shaped scaffold formed by six proteasome-COP9/CSN-initiation factor (PCI)-containing subunits. The PCI domains are essential for lid assembly, whereas the detailed molecular mechanisms remain elusive. Recent cryo-EM studies at near-atomic resolution provided invaluable information on the RP architecture in different functional states. Nevertheless, atomic resolution structural information on the RP is still limited, and deeper understanding of RP assembly mechanism requires further studies on the structures and interactions of individual subunits or subcomplexes. Herein we report the high-resolution NMR structures of the PCI-containing subunit Rpn9 from Saccharomyces cerevisiae. The 45-kDa protein contains an all-helical N-terminal domain and a C-terminal PCI domain linked via a semiflexible hinge. The N-terminal domain mediates interaction with the ubiquitin receptor Rpn10, whereas the PCI domain mediates interaction with the neighboring PCI subunit Rpn5. The Rpn9-Rpn5 interface highlights two structural motifs on the winged helix module forming a hydrophobic center surrounded by ionic pairs, which is a common pattern for all PCI-PCI interactions in the lid. The results suggest that divergence in surface composition among different PCI pairs may contribute to the modulation of lid assembly.

Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5-0.3 ppm for (13)C line widths and <0.5 ppm (15)N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via (1)H detected MAS NMR.

Navigating Membrane Protein Structure, Dynamics, and Energy Landscapes Using Spin Labeling and EPR Spectroscopy.

A detailed understanding of the functional mechanism of a protein entails the characterization of its energy landscape. Achieving this ambitious goal requires the integration of multiple approaches including determination of high-resolution crystal structures, uncovering conformational sampling under distinct biochemical conditions, characterizing the kinetics and thermodynamics of transitions between functional intermediates using spectroscopic techniques, and interpreting and harmonizing the data into novel computational models. With increasing sophistication in solution-based and ensemble-oriented biophysical approaches such as electron paramagnetic resonance (EPR) spectroscopy, atomic resolution structural information can be directly linked to conformational sampling in solution. Here, we detail how recent methodological and technological advances in EPR spectroscopy have contributed to the elucidation of membrane protein mechanisms. Furthermore, we aim to assist investigators interested in pursuing EPR studies by providing an introduction to the technique, a primer on experimental design, and a description of the practical considerations of the method toward generating high quality data.

Exploring the atomic structure and conformational flexibility of a 320 Å long engineered viral fiber using X-ray crystallography

Protein fibers are widespread in nature, but only a limited number of high-resolution structures have been determined experimentally. Unlike globular proteins, fibers are usually recalcitrant to form three-dimensional crystals, preventing single-crystal X-ray diffraction analysis. In the absence of three-dimensional crystals, X-ray fiber diffraction is a powerful tool to determine the internal symmetry of a fiber, but it rarely yields atomic resolution structural information on complex protein fibers. An 85-residue-long minimal coiled-coil repeat unit (MiCRU) was previously identified in the trimeric helical core of tail needle gp26, a fibrous protein emanating from the tail apparatus of the bacteriophage P22 virion. Here, evidence is provided that an MiCRU can be inserted in frame inside the gp26 helical core to generate a rationally extended fiber (gp26-2M) which, like gp26, retains a trimeric quaternary structure in solution. The 2.7 Å resolution crystal structure of this engineered fiber, which measures ∼320 Å in length and is only 20-35 Å wide, was determined. This structure, the longest for a trimeric protein fiber to be determined to such a high resolution, reveals the architecture of 22 consecutive trimerization heptads and provides a framework to decipher the structural determinants for protein fiber assembly, stability and flexibility.

Microcrystal manipulation with laser tweezers

X-ray crystallography is the method of choice to deduce atomic resolution structural information from macromolecules. In recent years, significant investments in structural genomics initiatives have been undertaken to automate all steps in X-ray crystallography from protein expression to structure solution. Robotic systems are widely used to prepare crystallization screens and change samples on synchrotron beamlines for macromolecular crystallography. The only remaining manual handling step is the transfer of the crystal from the mother liquor onto the crystal holder. Manual mounting is relatively straightforward for crystals with dimensions of >25 µm; however, this step is nontrivial for smaller crystals. The mounting of microcrystals is becoming increasingly important as advances in microfocus synchrotron beamlines now allow data collection from crystals with dimensions of only a few micrometres. To make optimal usage of these beamlines, new approaches have to be taken to facilitate and automate this last manual handling step. Optical tweezers, which are routinely used for the manipulation of micrometre-sized objects, have successfully been applied to sort and mount macromolecular crystals on newly designed crystal holders. Diffraction data from CPV type 1 polyhedrin microcrystals mounted with laser tweezers are presented.

Atomic-resolution structural information from scattering experiments on macromolecules in solution

The pair-distance distribution function (PDDF) contains all structural information probed in an elastic scattering experiment of macromolecular solutions. However, in small-angle x-ray scattering (SAXS) or small-angle neutron scattering (SANS) experiments only their Fourier transform is measured over a restricted range of scattering angles. We therefore developed a mathematically simple and computationally efficient method to calculate the PDDFs as well as accurate scattering intensities from molecular dynamics simulations. The calculated solution scattering intensities are in excellent agreement with SAXS and wide-angle x-ray scattering (WAXS) experiments for a series of proteins. The corresponding PDDFs are remarkably rich in features reporting on the detailed protein structure. Using an inverse Fourier transform method, most of these features can be recovered if scattering intensities are measured up to a momentum transfer of q≈2-3Å(-1). Our results establish that high-precision solution scattering experiments utilizing x-ray free-electron lasers and third generation synchrotron sources can resolve subnanometer structural detail, well beyond size, shape, and fold.

INstruct: a database of high-quality 3D structurally resolved protein interactome networks

INstruct is a database of high-quality, 3D, structurally resolved protein interactome networks in human and six model organisms. INstruct combines the scale of available high-quality binary protein interaction data with the specificity of atomic-resolution structural information derived from co-crystal evidence using a tested interaction interface inference method. Its web interface is designed to allow for flexible search based on standard and organism-specific protein and gene-naming conventions, visualization of protein architecture highlighting interaction interfaces and viewing and downloading custom 3D structurally resolved interactome datasets. INstruct is freely available on the web at http://instruct.yulab.org with all major browsers supported.