Cu(II) Binding Increases the Soluble Toxicity of Amyloidogenic Light Chains.

Light chain amyloidosis (AL) is caused by the aberrant overproduction of immunoglobulin light chains (LCs). The resulting abnormally high LC concentrations in blood lead to deposit formation in the heart and other target organs. Organ damage is caused not only by the accumulation of bulky amyloid deposits, but extensive clinical data indicate that circulating soluble LCs also exert cardiotoxic effects. The nematode C. elegans has been validated to recapitulate LC soluble toxicity in vivo, and in such a model a role for copper ions in increasing LC soluble toxicity has been reported. Here, we applied microscale thermophoresis, isothermal calorimetry and thermal melting to demonstrate the specific binding of Cu2+ to the variable domain of amyloidogenic H7 with a sub-micromolar affinity. Histidine residues present in the LC sequence are not involved in the binding, and yet their mutation to Ala reduces the soluble toxicity of H7. Copper ions bind to and destabilize the variable domains and induce a limited stabilization in this domain. In summary, the data reported here, elucidate the biochemical bases of the Cu2+-induced toxicity; moreover, they also show that copper binding is just one of the several biochemical traits contributing to LC soluble in vivo toxicity.

Building Structural Models of a Whole Mycoplasma Cell.

Building structural models of entire cells has been a long-standing cross-discipline challenge for the research community, as it requires an unprecedented level of integration between multiple sources of biological data and enhanced methods for computational modeling and visualization. Here, we present the first 3D structural models of an entire Mycoplasma genitalium (MG) cell, built using the CellPACK suite of computational modeling tools. Our model recapitulates the data described in recent whole-cell system biology simulations and provides a structural representation for all MG proteins, DNA and RNA molecules, obtained by combining experimental and homology-modeled structures and lattice-based models of the genome. We establish a framework for gathering, curating and evaluating these structures, exposing current weaknesses of modeling methods and the boundaries of MG structural knowledge, and visualization methods to explore functional characteristics of the genome and proteome. We compare two approaches for data gathering, a manually-curated workflow and an automated workflow that uses homologous structures, both of which are appropriate for the analysis of mesoscale properties such as crowding and volume occupancy. Analysis of model quality provides estimates of the regularization that will be required when these models are used as starting points for atomic molecular dynamics simulations.

CellPAINT: Turnkey Illustration of Molecular Cell Biology.

CellPAINT is an interactive digital tool that allows non-expert users to create illustrations of the molecular structure of cells and viruses. We present a new release with several key enhancements, including the ability to generate custom ingredients from structure information in the Protein Data Bank, and interaction, grouping, and locking functions that streamline the creation of assemblies and illustration of large, complex scenes. An example of CellPAINT as a tool for hypothesis generation in the interpretation of cryoelectron tomograms is presented. CellPAINT is freely available at http://ccsb.scripps.edu/cellpaint.

Moltemplate: A Tool for Coarse-Grained Modeling of Complex Biological Matter and Soft Condensed Matter Physics.

Coarse-grained models have long been considered indispensable tools in the investigation of biomolecular dynamics and assembly. However, the process of simulating such models is arduous because unconventional force fields and particle attributes are often needed, and some systems are not in thermal equilibrium. Although modern molecular dynamics programs are highly adaptable, software designed for preparing all-atom simulations typically makes restrictive assumptions about the nature of the particles and the forces acting on them. Consequently, the use of coarse-grained models has remained challenging. Moltemplate is a file format for storing coarse-grained molecular models and the forces that act on them, as well as a program that converts moltemplate files into input files for LAMMPS, a popular molecular dynamics engine. Moltemplate has broad scope and an emphasis on generality. It accommodates new kinds of forces as they are developed for LAMMPS, making moltemplate a popular tool with thousands of users in computational chemistry, materials science, and structural biology. To demonstrate its wide functionality, we provide examples of using moltemplate to prepare simulations of fluids using many-body forces, coarse-grained organic semiconductors, and the motor-driven supercoiling and condensation of an entire bacterial chromosome.

Modeling in the Time of COVID-19: Statistical and Rule-based Mesoscale Models.

We present a new technique for the rapid modeling and construction of scientifically accurate mesoscale biological models. The resulting 3D models are based on a few 2D microscopy scans and the latest knowledge available about the biological entity, represented as a set of geometric relationships. Our new visual-programming technique is based on statistical and rule-based modeling approaches that are rapid to author, fast to construct, and easy to revise. From a few 2D microscopy scans, we determine the statistical properties of various structural aspects, such as the outer membrane shape, the spatial properties, and the distribution characteristics of the macromolecular elements on the membrane. This information is utilized in the construction of the 3D model. Once all the imaging evidence is incorporated into the model, additional information can be incorporated by interactively defining the rules that spatially characterize the rest of the biological entity, such as mutual interactions among macromolecules, and their distances and orientations relative to other structures. These rules are defined through an intuitive 3D interactive visualization as a visual-programming feedback loop. We demonstrate the applicability of our approach on a use case of the modeling procedure of the SARS-CoV-2 virion ultrastructure. This atomistic model, which we present here, can steer biological research to new promising directions in our efforts to fight the spread of the virus.

Inherent Biophysical Properties Modulate the Toxicity of Soluble Amyloidogenic Light Chains.

In light chain amyloidosis (AL), fibrillar deposition of monoclonal immunoglobulin light chains (LCs) in vital organs, such as heart, is associated with their severe dysfunction. In addition to the cellular damage caused by fibril deposition, direct toxicity of soluble prefibrillar amyloidogenic proteins has been reported, in particular, for cardiotoxicity. However, the molecular bases of proteotoxicity by soluble LCs have not been clarified. Here, to address this issue, we rationally engineered the amino acid sequence of the highly cardiotoxic LC H6 by introducing three residue mutations, designed to reduce the dynamics of its native state. The resulting mutant (mH6) is less toxic than its parent H6 to human cardiac fibroblasts and C. elegans. The high sequence and structural similarity, together with the different toxicity, make H6 and its non-toxic designed variant mH6 a test case to shed light on the molecular properties underlying soluble toxicity. Our comparative structural and biochemical study of H6 and mH6 shows closely matching crystal structures, whereas spectroscopic data and limited proteolysis indicate that H6 displays poorly cooperative fold, higher flexibility, and kinetic instability, and a higher dynamic state in its native fold. Taken together, the results of this study show a strong correlation between the overall conformational properties of the native fold and the proteotoxicity of cardiotropic LCs.

Cryo-EM structure of cardiac amyloid fibrils from an immunoglobulin light chain AL amyloidosis patient.

Systemic light chain amyloidosis (AL)  is a life-threatening disease caused by aggregation and deposition of monoclonal immunoglobulin light chains (LC) in target organs. Severity of heart involvement is the most important factor determining prognosis. Here, we report the 4.0 Å resolution cryo-electron microscopy map and molecular model of amyloid fibrils extracted from the heart of an AL amyloidosis patient with severe amyloid cardiomyopathy. The helical fibrils are composed of a single protofilament, showing typical 4.9 Å stacking and cross-ß architecture. Two distinct polypeptide stretches (total of 77 residues) from the LC variable domain (Vl) fit the fibril density. Despite Vl high sequence variability, residues stabilizing the fibril core are conserved through different cardiotoxic Vl, highlighting structural motifs that may be common to misfolding-prone LCs. Our data shed light on the architecture of LC amyloids, correlate amino acid sequences with fibril assembly, providing the grounds for development of innovative medicines.

Structures of NHBA elucidate a broadly conserved epitope identified by a vaccine induced antibody.

Neisserial heparin binding antigen (NHBA) is one of three main recombinant protein antigens in 4CMenB, a vaccine for the prevention of invasive meningococcal disease caused by Neisseria meningitidis serogroup B. NHBA is a surface-exposed lipoprotein composed of a predicted disordered N-terminal region, an arginine-rich region that binds heparin, and a C-terminal domain that folds as an anti-parallel ß-barrel and that upon release after cleavage by human proteases alters endothelial permeability. NHBA induces bactericidal antibodies in humans, and NHBA-specific antibodies elicited by the 4CMenB vaccine contribute to serum bactericidal activity, the correlate of protection. To better understand the structural bases of the human antibody response to 4CMenB vaccination and to inform antigen design, we used X-ray crystallography to elucidate the structures of two C-terminal fragments of NHBA, either alone or in complex with the Fab derived from the vaccine-elicited human monoclonal antibody 5H2, and the structure of the unbound Fab 5H2. The structures reveal details on the interaction between an N-terminal ß-hairpin fragment and the ß-barrel, and explain how NHBA is capable of generating cross-reactive antibodies through an extensive conserved conformational epitope that covers the entire C-terminal face of the ß-barrel. By providing new structural information on a vaccine antigen and on the human immune response to vaccination, these results deepen our molecular understanding of 4CMenB, and might also aid future vaccine design projects.

Concurrent structural and biophysical traits link with immunoglobulin light chains amyloid propensity.

Light chain amyloidosis (AL), the most common systemic amyloidosis, is caused by the overproduction and the aggregation of monoclonal immunoglobulin light chains (LC) in target organs. Due to genetic rearrangement and somatic hypermutation, virtually, each AL patient presents a different amyloidogenic LC. Because of such complexity, the fine molecular determinants of LC aggregation propensity and proteotoxicity are, to date, unclear; significantly, their decoding requires investigating large sets of cases. Aiming to achieve generalizable observations, we systematically characterised a pool of thirteen sequence-diverse full length LCs. Eight amyloidogenic LCs were selected as responsible for severe cardiac symptoms in patients; five non-amyloidogenic LCs were isolated from patients affected by multiple myeloma. Our comprehensive approach (consisting of spectroscopic techniques, limited proteolysis, and X-ray crystallography) shows that low fold stability and high protein dynamics correlate with amyloidogenic LCs, while hydrophobicity, structural rearrangements and nature of the LC dimeric association interface (as observed in seven crystal structures here presented) do not appear to play a significant role in defining amyloid propensity. Based on the structural and biophysical data, our results highlight shared properties driving LC amyloid propensity, and these data will be instrumental for the design of synthetic inhibitors of LC aggregation.

Crystal structures of human Fabs targeting the Bexsero meningococcal vaccine antigen NHBA.

Neisserial heparin-binding antigen (NHBA) is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero. As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero, the crystal structures of two fragment antigen-binding domains (Fabs) isolated from human monoclonal antibodies targeting NHBA were determined. Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs, these structures provide broad insights into the NHBA epitopes recognized by the human immune system. As expected, these Fabs also show remarkable structural conservation, as shown by a structural comparison of 15 structures of apo Fab 10C3 which were obtained from crystals grown in different crystallization conditions and were solved while searching for a complex with a bound NHBA fragment or epitope peptide. This study also provides indirect evidence for the intrinsically disordered nature of two N-terminal regions of NHBA.

Crystal structure of FhuD at 1.6†Šresolution: a ferrichrome-binding protein from the animal and human pathogen Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is a leading cause of disease in dogs, and zoonosis causes human infections. Methicillin-resistant S. pseudintermedius strains are emerging, resembling the global health threat of S. aureus. Therefore, it is increasingly important to characterize potential targets for intervention against S. pseudintermedius. Here, FhuD, an S. pseudintermedius surface lipoprotein implicated in iron uptake, was characterized. It was found that FhuD bound ferrichrome in an iron-dependent manner, which increased the thermostability of FhuD by >15 °C. The crystal structure of ferrichrome-free FhuD was determined via molecular replacement at 1.6 Å resolution. FhuD exhibits the class III solute-binding protein (SBP) fold, with a ligand-binding cavity between the N- and C-terminal lobes, which is here occupied by a PEG molecule. The two lobes of FhuD were oriented in a closed conformation. These results provide the first detailed structural characterization of FhuD, a potential therapeutic target of S. pseudintermedius.