Structural Biology Approaches Research Articles

Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals.

The crystallization of recombinant proteins in living cells is an exciting new approach in structural biology. Recent success has highlighted the need for fast and efficient diffraction data collection, optimally directly exposing intact crystal-containing cells to the X-ray beam, thus protecting the in cellulo crystals from environmental challenges. Serial femtosecond crystallography (SFX) at free-electron lasers (XFELs) allows the collection of detectable diffraction even from tiny protein crystals, but requires very fast sample exchange to utilize each XFEL pulse. Here, an efficient approach is presented for high-resolution structure elucidation using serial femtosecond in cellulo diffraction of micometre-sized crystals of the protein HEX-1 from the fungus Neurospora crassa on a fixed target. Employing the fast and highly accurate Roadrunner II translation-stage system allowed efficient raster scanning of the pores of micro-patterned, single-crystalline silicon chips loaded with living, crystal-containing insect cells. Compared with liquid-jet and LCP injection systems, the increased hit rates of up to 30% and reduced background scattering enabled elucidation of the HEX-1 structure. Using diffraction data from only a single chip collected within 12 min at the Linac Coherent Light Source, a 1.8 Å resolution structure was obtained with significantly reduced sample consumption compared with previous SFX experiments using liquid-jet injection. This HEX-1 structure is almost superimposable with that previously determined using synchrotron radiation from single HEX-1 crystals grown by sitting-drop vapour diffusion, validating the approach. This study demonstrates that fixed-target SFX using micro-patterned silicon chips is ideally suited for efficient in cellulo diffraction data collection using living, crystal-containing cells, and offers huge potential for the straightforward structure elucidation of proteins that form intracellular crystals at both XFELs and synchrotron sources.

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1

Talin (TLN1) is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behavior of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronized phosphorylation of a large number of protein targets. CDK1 activity maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesion that are required for successful mitosis. Using a combination of biochemical, structural, and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8. Talin also contains a consensus CDK1 phosphorylation motif centered on S1589, a site shown to be phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of the cytoskeletal adaptor KANK and weakened the response of this region to force as measured by single molecule stretching, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator CDK1 to the primary integrin effector talin represents a coupling of cell proliferation and cell adhesion machineries and thereby indicates a mechanism by which the microenvironment can control cell division in multicellular organisms.

Abstract P-30: Structure of the 80S Ribosome From Candida Albicans Revealed by Integrative Structural Biology Approach

Background: The fungus Candida albicans is one of the most common fungal human pathogens, causing numerous mucocutaneous infections. About 80% of the world population is colonized with this pathogen, and in many cases, it does not reveal its pathogenic properties. However in the events when the suppression of normal bacterial flora occurs, e.g. as a consequence of chemotherapy or antibiotics treatment, multi-resistant strains of Candida albicans can rapidly colonize the host organism and prevent the normal flora recovery, which in turn can trigger many other diseases especially in the case of immunocompromised patients. Methods: We used an integrated structural biology approach based on the single-particle cryo-EM reconstruction and macromolecule X-ray crystallography. Results: One of the most promising targets for antibiotic action is a protein synthesis apparatus and ribosomes in the cell. In this study, we characterized structurally the protein synthesis machinery of Candida albicans using the state-of-the-art techniques of single-particle Cryo-electron microscopy and macromolecular X-ray crystallography. Conclusion: We obtained the 2.4 Å resolution structure of the 80S ribosome from Candida albicans and the 4.2 Å resolution structure of the vacant C. albicans ribosome by X-ray crystallography. We believe that this study will shed light on mechanisms of antimicrobial resistance in C. albicans and improve candidiasis treatment in the future.

B2bTools: online predictions for protein biophysical features and their conservation.

We provide integrated protein sequence-based predictions via https://bio2byte.be/b2btools/. The aim of our predictions is to identify the biophysical behaviour or features of proteins that are not readily captured by structural biology and/or molecular dynamics approaches. Upload of a FASTA file or text input of a sequence provides integrated predictions from DynaMine backbone and side-chain dynamics, conformational propensities, and derived EFoldMine early folding, DisoMine disorder, and Agmata β-sheet aggregation. These predictions, several of which were previously not available online, capture ‘emergent’ properties of proteins, i.e. the inherent biophysical propensities encoded in their sequence, rather than context-dependent behaviour (e.g. final folded state). In addition, upload of a multiple sequence alignment (MSA) in a variety of formats enables exploration of the biophysical variation observed in homologous proteins. The associated plots indicate the biophysical limits of functionally relevant protein behaviour, with unusual residues flagged by a Gaussian mixture model analysis. The prediction results are available as JSON or CSV files and directly accessible via an API. Online visualisation is available as interactive plots, with brief explanations and tutorial pages included. The server and API employ an email-free token-based system that can be used to anonymously access previously generated results.

Cryo-EM: A new dawn in thyroid biology

The thyroid gland accumulates the rare dietary element iodine and incorporates it into iodinated thyroid hormones, utilising several tightly regulated reactions and molecular mechanisms. Thyroid hormones are essential in vertebrates and play a central role in many biological processes, such as development, thermogenesis and growth. The control of these functions is exerted through the binding of hormones to nuclear thyroid hormone receptors that rule the transcription of numerous metabolic genes. Over the last 50 years, thyroid biology has been studied extensively at the cellular and organismal levels, revealing its multiple clinical implications, yet, a complete molecular understanding is still lacking. This includes the atomic structures of crucial pathway components that would be needed to elucidate molecular mechanisms. Here we review the currently known protein structures involved in thyroid hormone synthesis, regulation, transport, and actions. We also highlight targets for future investigations that will significantly benefit from recent advances in macromolecular structure determination by electron cryo-microscopy (cryo-EM). As an example, we demonstrate how cryo-EM was crucial to obtain the structure of the large thyroid hormone precursor protein, thyroglobulin. We discuss modern cryo-EM compared to other structure determination methods and how an integrated structural and cell biological approach will help filling the molecular knowledge gap in our understanding of thyroid hormone metabolism. Together with clinical, cellular and high-throughput 'omics' studies, atomic structures of thyroid components will provide an important framework to map disease mutations and to interpret and predict thyroid phenotypes.

PXR and Metabolism

Pregnane X receptor (PXR) is a nuclear receptor initially characterized as a xenobiotic-sensing receptor that transcriptionally induces drug-metabolizing enzymes and transporters to control the disposition of xenobiotics. Emerging data reveal PXR's broader roles in regulating glucose and lipid metabolism to control metabolic homeostasis. In addition, there has been evidence of crosstalk among the PXR-regulated metabolism of drugs, lipids, and glucose. Such crosstalk might play roles in the development of diseases such as diabetes and cancer, and the therapeutic and toxic effects of drugs. The activity of PXR can be modulated by many structurally diverse chemicals. However, potent and selective chemical modulators of PXR was lacking. By using chemical biology, structural biology, and pharmacology approaches, we have developed a series of potent, selective and structurally related PXR modulators (i.e., agonists, antagonists, and inverse agonists). We discovered that subtle structural modifications of the compounds, or a mutation within the PXR ligand-binding domain can convert an antagonist to an agonist. While ligand binding affinity does not predict PXR's cellular activity, there is a correlation between ligand-induced positioning of the activation function 2 (AF-2) helix and PXR's transcriptional activity. We are using these novel chemical probes to investigate the broad metabolic function of PXR, with a goal to use them to develop therapies for PXR-regulated diseases.

Histone Recognition by the BDP1 Bromodomain of Plasmodium falciparum

Bromodomains are evolutionarily conserved protein modules that specifically recognize acetylated lysine residues on histone tails of the nucleosome. During the red-blood-cell-stage of infection with Plasmodium falciparum, the parasite undergoes repeated rounds of replication, egress, and invasion. Erythrocyte invasion involves specific interactions between host cell receptors and parasite ligands and coordinated expression of genes specific to this step of the life cycle. A parasite-specific protein known as bromodomain protein 1 (PfBDP1) binds to chromatin at the transcriptional start sites of invasion-related genes and directly controls their expression. Conditional PfBDP1 knockdown causes a dramatic defect in parasite invasion and growth, and results in transcriptional down-regulation of multiple invasion-related genes at a time point critical for invasion. Conversely, PfBDP1 overexpression enhances expression of these same invasion-related genes. PfBDP1 has been shown to interact with acetylated histone H3 and a second bromodomain protein, PfBDP2, suggesting a potential mechanism for gene recognition and control. Since PfBDP1 critically coordinates expression of invasion genes, targeting PfBDP1 could be an invaluable tool in mitigating malaria infections. The domain architecture of PfBDP1 includes a single C-terminal bromodomain and several ankyrin repeats. PfBDP1 is thought to act by tethering a transcriptional activator complex to acetylated histone H3 to control genes required for parasite invasion. We hypothesized that the bromodomain is crucial for this function of PfBDP1 via recognition of acetylated lysine modifications. We carried out several histone binding assays to identify novel histone interactions the of PfBDP1 bromodomain. We also utilized a combination of structural biology and biophysical approaches to identify amino acid residues critical for ligand coordination. The result of this study will provide molecular details about the recognition of acetylated histone ligands by the PfBDP1 bromodomain and shed light on how this domain recruits the PfBDP1-PfBDP2 complex to chromatin. Importantly, these results may improve the development of new therapeutics to treat malaria

Structure of the ATP‐free Mre11‐Rad50 DNA damage repair complex bound to DNA substrates

DNA double strand breaks, where both strands of the double-stranded DNA are broken, are among the most harmful forms of DNA damage because an intact template is not present for repair. The Mre11-Rad50–Nbs1 (Xrs2 in yeast) complex plays a central and critical role in the detection and repair of DSBs and is conserved in all kingdoms of life, as Mre11Rad50 (MR) in prokaryotes and as MRN/X in eukaryotes. Although there are some structural models available for MR complex bound to DNA substrates, there are limitations in these structures. Therefore, our goal is to examine the MR complex bound to different DNA substrates using an integrated structural biology approach. Using uniformly deuterated, 13 CH -methyl labeled samples, we measured distance restraints for the ~120 kDa P. furiosus MR complex bound to spin-labeled double-stranded or single-stranded DNA substrates 3 via paramagnetic relaxation enhancements (PREs). Additional distance and global restraints were measured from Luminescence Resonance Energy Transfer (LRET) and Small Angle X-ray Scatter (SAXS) experiments. These data then served as restraints for calculating structural models using multi-body docking implemented in HADDOCK. Our models revealed novel modes of DNA binding for the ATP-free MR complex and provide the first depiction of a single-stranded DNA-bound complex. We tested our models through biochemical activity assays to determine the effect of disruption of key interactions, via mutation, in our models.

A cross-neutralizing antibody between HIV-1 and influenza virus.

Incessant antigenic evolution enables the persistence and spread of influenza virus in the human population. As the principal target of the immune response, the hemagglutinin (HA) surface antigen on influenza viruses continuously acquires and replaces N-linked glycosylation sites to shield immunogenic protein epitopes using host-derived glycans. Anti-glycan antibodies, such as 2G12, target the HIV-1 envelope protein (Env), which is even more extensively glycosylated and contains under-processed oligomannose-type clusters on its dense glycan shield. Here, we illustrate that 2G12 can also neutralize human seasonal influenza A H3N2 viruses that have evolved to present similar oligomannose-type clusters on their HAs from around 20 years after the 1968 pandemic. Using structural biology and mass spectrometric approaches, we find that two N-glycosylation sites close to the receptor binding site (RBS) on influenza hemagglutinin represent the oligomannose cluster recognized by 2G12. One of these glycan sites is highly conserved in all human H3N2 strains and the other emerged during virus evolution. These two N-glycosylation sites have also become crucial for fitness of recent H3N2 strains. These findings shed light on the evolution of the glycan shield on influenza virus and suggest 2G12-like antibodies can potentially act as broad neutralizers to target human enveloped viruses.

Crosslinking mass spectrometry: A link between structural biology and systems biology.

Protein structure underpins functional roles in all biological processes; therefore, improved understanding of protein structures is of fundamental importance in nearly all biological and biomedical research areas. Traditional techniques such as X-ray crystallography and more recently, cryo-EM, can reveal structural features on isolated proteins/protein complexes at atomic resolution level and have become indispensable tools for structural biology. Crosslinking mass spectrometry (XL-MS), on the other hand, is an emerging technique capable of capturing transient and dynamic information on protein interactions and assemblies in their native environment. The combination of XL-MS with traditional techniques holds potential for bridging the gap between structural biology and systems biology approaches. Such a combination will enable visualization of protein structures and interactions within the crowded macromolecular environment in living systems that can dramatically increase understanding of biological functions. In this review, we first discuss general strategies of XL-MS and then survey recent examples to show how qualitative and quantitative XL-MS studies can be integrated with available protein structural data to better understand biological function at systems level.

Discovery of a Novel CIP2A Variant (NOCIVA) with Clinical Relevance in Predicting TKI Resistance in Myeloid Leukemias.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein that inhibits the tumor suppressor PP2A-B56α. However, CIP2A mRNA variants remain uncharacterized. Here, we report the discovery of a CIP2A splicing variant, novel CIP2A variant (NOCIVA). Characterization of CIP2A variants was performed by both 3' and 5' rapid amplification of cDNA ends from cancer cells. The function of NOCIVA was assessed by structural and molecular biology approaches. Its clinical relevance was studied in an acute myeloid leukemia (AML) patient cohort and two independent chronic myeloid leukemia (CML) cohorts. NOCIVA contains CIP2A exons 1 to 13 fused to 349 nucleotides from CIP2A intron 13. Intriguingly, the first 39 nucleotides of the NOCIVA-specific sequence are in the coding frame with exon 13 of CIP2A and code for a 13-amino acid peptide tail nonhomologous to any known human protein sequence. Therefore, NOCIVA translates to a unique human protein. NOCIVA retains the capacity to bind to B56α, but, whereas CIP2A is predominantly a cytoplasmic protein, NOCIVA translocates to the nucleus. Indicative of prevalent alternative splicing from CIP2A to NOCIVA in myeloid malignancies, AML and CML patient samples overexpress NOCIVA, but not CIP2A mRNA. In AML, a high NOCIVA/CIP2A mRNA expression ratio is a marker for adverse overall survival. In CML, high NOCIVA expression is associated with inferior event-free survival among imatinib-treated patients, but not among patients treated with dasatinib or nilotinib. We discovered a novel variant of the oncoprotein CIP2A and its clinical relevance in predicting tyrosine kinase inhibitor therapy resistance in myeloid leukemias.

Structural biology of coronavirus ion channels

Viral infection compromises specific organelles of the cell and readdresses its functional resources to satisfy the needs of the invading body. Around 70% of the coronavirus positive-sense single-stranded RNA encodes proteins involved in replication, and these viruses essentially take over the biosynthetic and transport mechanisms to ensure the efficient replication of their genome and trafficking of their virions. Some coronaviruses encode genes for ion-channel proteins - the envelope protein E (orf4a), orf3a and orf8 - which they successfully employ to take control of the endoplasmic reticulum-Golgi complex intermediate compartment or ERGIC. The E protein, which is one of the four structural proteins of SARS-CoV-2 and other coronaviruses, assembles its transmembrane protomers into homopentameric channels with mild cationic selectivity. Orf3a forms homodimers and homotetramers. Both carry a PDZ-binding domain, lending them the versatility to interact with more than 400 target proteins in infected host cells. Orf8 is a very short 29-amino-acid single-passage transmembrane peptide that forms cation-selective channels when assembled in lipid bilayers. This review addresses the contribution of biophysical and structural biology approaches that unravel different facets of coronavirus ion channels, their effects on the cellular machinery of infected cells and some structure-functional correlations with ion channels of higher organisms.

Gas-phase ion/ion chemistry for structurally sensitive probes of gaseous protein ion structure: Electrostatic and electrostatic to covalent cross-linking

Intramolecular interactions within a protein are key in maintaining protein tertiary structure and understanding how proteins function. Ion mobility-mass spectrometry (IM-MS) has become a widely used approach in structural biology since it provides rapid measurements of collision cross sections (CCS), which inform on the gas-phase conformation of the biomolecule under study. Gas-phase ion/ion reactions target amino acid residues with specific chemical properties and the modified sites can be identified by MS. In this study, electrostatically reactive, gas-phase ion/ion chemistry and IM-MS are combined to characterize the structural changes between ubiquitin electrosprayed from aqueous and denaturing conditions. The electrostatic attachment of sulfo-NHS acetate to ubiquitin via ion/ion reactions and fragmentation by electron-capture dissociation (ECD) provide the identification of the most accessible protonated sites within ubiquitin as the sulfonate group forms an electrostatic complex with accessible protonated side chains. The protonated sites identified by ECD from the different solution conditions are distinct and, in some cases, reflect the disruption of interactions such as salt bridges that maintain the native protein structure. This agrees with previously published literature demonstrating that a high methanol concentration at low pH causes the structure of ubiquitin to change from a native (N) state to a more elongated A state. Results using gas-phase, electrostatic cross-linking reagents also point to similar structural changes and further confirm the role of methanol and acid in favoring a more unfolded conformation. Since cross-linking reagents have a distance constraint for the two reactive sites, the data is valuable in guiding computational structures generated by molecular dynamics. The research presented here describes a promising strategy that can detect subtle changes in the local environment of targeted amino acid residues to inform on changes in the overall protein structure.

Attenuation of nociceptive and paclitaxel-induced neuropathic pain by targeting inflammatory, CGRP and substance P signaling using 3-Hydroxyflavone

Paclitaxel is an anti-microtubule agent, most widely used chemotherapeutic agent for the treatment of malignant solid tumors. However, it is associated with some severe side effects including painful neurotoxicity with reporting of neuropathic pain and sensory abnormalities by patients during and after paclitaxel therapy. Peripheral neuropathy was induced by the administration of paclitaxel (4 mg/kg on days 1, 3, 5, and 7). In this study, the anti-nociceptive and anti-inflammatory propensity of 3-Hydroxyflavone (3HF) in mice and the preventive effect of 3HF against paclitaxel-induced peripheral neuropathy in Sprague Dawley (SD) rats were investigated. Moreover, tactile and cold allodynia, thermal and tail immersion hyperalgesia, and effects on motor-coordination were also evaluated. Furthermore, the expression of proinflammatory cytokines i.e. Calcitonin gene-related peptide (CGRP), and Substance P from the spinal cord was examined through RT-PCR. Additionally, a computational structural biology approach was applied to search the potential therapeutic targets and to predict the binding mechanism of 3HF.Treatment of 3HF alleviated the nociceptive pain, paw edema, development of tactile and cold allodynia, and hyperalgesia. Similarly, treatment with 3HF suppressed the paclitaxel-induced increase in mRNA expression of several inflammatory cytokines including tumor necrosis factor -α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), CGRP, and Substance P. However, the daily treatment of 3HF did not affect the motor behaviors of rats. The inhibitory mechanism of 3HF in neuropathic pain is predicted with extensive computational bioinformatics approach which indicates that the 3HF effectively interacts with the binding domains of Nuclear factor-kappa B (NF-κB), CGRP receptor and the receptor of Substance P to exert its inhibitory activities. However, the computationally predicted binding affinities revealed that the potential of binding of the compound with Substance P receptor (Neurokinin 1 receptor) is higher than the other receptors; there NK1R could be the most possible binding target of 3HF. These findings indicate that 3HF has anti-nociceptive, anti-inflammatory, and anti-neuropathic pain effects against paclitaxel-induced neuropathic pain.

Sarcomeric Protein Fatz Forms a Tight Fuzzy Complex with α-Actinin and Phase-Separates in Vitro

In sarcomeres, α-actinin crosslinks actin filaments and anchors them to the Z-disk. FATZ proteins (filamin, α-actinin and telethonin binding proteins of the Z-disk) interact with α-actinin and five other core Z-disk proteins, contributing to myofibril assembly and maintenance as a protein interaction hub. However, the molecular mechanism behind the FATZ scaffolding function is unknown. Here, we investigated the interaction of FATZ-1 with α-actinin-2 using an integrative structural biology approach. We provide the first structure of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which alone is best described as a dynamic ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2, and propose a molecular mechanism for interaction via molecular recognition elements and additional transient binding sites, leading to differential affinities of two FATZ-1 molecules for the α-actinin-2 dimer and a polar architecture of the assembly. in combination with FATZ-1 multivalent scaffolding function, this assembly is likely to organise interaction partners and stabilise the preferential orientation of α-actinin-2 in Z-disk.Finally, we uncover the ability of FATZ-1 to phase-separate and form biomolecular condensates with α-actinin-2, suggesting an intriguing hypothesis of FATZ proteins as an interaction hub for Z-disk partners through membrane-less compartmentalization at initial stages of myofibrillogenesis.

Structure of the ATP-Free Mre11-Rad50 DNA Damage Repair Complex Bound to DNA Substrates

DNA double strand breaks, where both strands of the double-stranded DNA are broken, are among the most harmful forms of DNA damage because an intact template is not present for repair. The Mre11-Rad50–Nbs1 (Xrs2 in yeast) complex plays a central and critical role in the detection and repair of DSBs and is conserved in all kingdoms of life, as Mre11- Rad50 (MR) in prokaryotes and as MRN/X in eukaryotes. Although there are some structural models available for MR complex bound to DNA substrates, there are limitations in these structures. Therefore, our goal is to examine the MR complex bound to different DNA substrates using an integrated structural biology approach. Using uniformly deuterated, 13CH3-methyl labeled samples, we measured distance restraints for the ∼120 kDa P. furiosus MR complex bound to spin-labeled double-stranded or single-stranded DNA substrates via paramagnetic relaxation enhancements (PREs). Additional distance and global restraints were measured from Luminescence Resonance Energy Transfer (LRET) and Small Angle X-ray Scatter (SAXS) experiments. These data then served as restraints for calculating structural models using multi-body docking implemented in HADDOCK. Our models revealed novel modes of DNA binding for the ATP-free MR complex and provide the first depiction of a single-stranded DNA-bound complex. We tested our models through biochemical activity assays to determine the effect of disruption of key interactions, via mutation, in our models.

The solution structures of higher-order human telomere G-quadruplex multimers.

Human telomeres contain the repeat DNA sequence 5′-d(TTAGGG), with duplex regions that are several kilobases long terminating in a 3′ single-stranded overhang. The structure of the single-stranded overhang is not known with certainty, with disparate models proposed in the literature. We report here the results of an integrated structural biology approach that combines small-angle X-ray scattering, circular dichroism (CD), analytical ultracentrifugation, size-exclusion column chromatography and molecular dynamics simulations that provide the most detailed characterization to date of the structure of the telomeric overhang. We find that the single-stranded sequences 5′-d(TTAGGG)n, with n = 8, 12 and 16, fold into multimeric structures containing the maximal number (2, 3 and 4, respectively) of contiguous G4 units with no long gaps between units. The G4 units are a mixture of hybrid-1 and hybrid-2 conformers. In the multimeric structures, G4 units interact, at least transiently, at the interfaces between units to produce distinctive CD signatures. Global fitting of our hydrodynamic and scattering data to a worm-like chain (WLC) model indicates that these multimeric G4 structures are semi-flexible, with a persistence length of ∼34 Å. Investigations of its flexibility using MD simulations reveal stacking, unstacking, and coiling movements, which yield unique sites for drug targeting.

The structural biology of crystallin aggregation: challenges and outlook.

Crystallin aggregation is characterized by light scattering of large molecular aggregates due to their phase separation in the lens. Low-resolution biophysical studies using multiple techniques have characterized the folding, stability, binding, and aggregation of crystallins in the past but with limited access to their structure, dynamics, and interactions. In this Viewpoint, three schools of experimental structural biology, that is, X-ray crystallography, solution and solid-state NMR spectroscopy, and cryo-electron microscopy, combine to provide atomic resolution details of native crystallins, soluble oligomers, and insoluble amyloid fibrils and amorphous aggregates. Computational structural biology provides additional details on crystallin dynamics and the crucial intercrystallin interactions in these events. Our current understanding of the diverse structural biology of crystallins is consistent with multiple pathways of protein aggregation for different structural intermediates. This Viewpoint combines our efforts with those of others to elucidate the recent progress in these high-resolution studies and proposes an integrated structural biology approach to resolve the complex lens interactome. Overall, I discuss the outstanding questions and evaluate the experimental and theoretical caveats in the field.

Parainfluenza virus entry at the onset of infection.

Parainfluenza viruses, members of the enveloped, negative-sense, single stranded RNA Paramyxoviridae family, impact global child health as the cause of significant lower respiratory tract infections. Parainfluenza viruses enter cells by fusing directly at the cell surface membrane. How this fusion occurs via the coordinated efforts of the two molecules that comprise the viral surface fusion complex, and how these efforts may be blocked, are the subjects of this chapter. The receptor binding protein of parainfluenza forms a complex with the fusion protein of the virus, remaining stably associated until a receptor is reached. At that point, the receptor binding protein actively triggers the fusion protein to undergo a series of transitions that ultimately lead to membrane fusion and viral entry. In recent years it has become possible to examine this remarkable process on the surface of viral particles and to begin to understand the steps in the transition of this molecular machine, using a structural biology approach. Understanding the steps in entry leads to several possible strategies to prevent fusion and inhibit infection.

Functional implications of MIR domains in protein O-mannosylation.

Protein O-mannosyltransferases (PMTs) represent a conserved family of multispanning endoplasmic reticulum membrane proteins involved in glycosylation of S/T-rich protein substrates and unfolded proteins. PMTs work as dimers and contain a luminal MIR domain with a β-trefoil fold, which is susceptive for missense mutations causing α-dystroglycanopathies in humans. Here, we analyze PMT-MIR domains by an integrated structural biology approach using X-ray crystallography and NMR spectroscopy and evaluate their role in PMT function in vivo. We determine Pmt2- and Pmt3-MIR domain structures and identify two conserved mannose-binding sites, which are consistent with general β-trefoil carbohydrate-binding sites (α, β), and also a unique PMT2-subfamily exposed FKR motif. We show that conserved residues in site α influence enzyme processivity of the Pmt1-Pmt2 heterodimer in vivo. Integration of the data into the context of a Pmt1-Pmt2 structure and comparison with homologous β-trefoil - carbohydrate complexes allows for a functional description of MIR domains in protein O-mannosylation.