Structure of a collagen VI α3 chain VWA domain array: adaptability and functional implications of myopathy causing mutations.

Collagen VI is a ubiquitous heterotrimeric protein of the extracellular matrix (ECM) that plays an essential role in the proper maintenance of skeletal muscle. Mutations in collagen VI lead to a spectrum of congenital myopathies, from the mild Bethlem myopathy to the severe Ullrich congenital muscular dystrophy. Collagen VI contains only a short triple helix and consists primarily of von Willebrand factor type A (VWA) domains, protein-protein interaction modules found in a range of ECM proteins. Disease-causing mutations occur commonly in the VWA domains, and the second VWA domain of the α3 chain, the N2 domain, harbors several such mutations. Here, we investigate structure-function relationships of the N2 mutations to shed light on their possible myopathy mechanisms. We determined the X-ray crystal structure of N2, combined with monitoring secretion efficiency in cell culture of selected N2 single-domain mutants, finding that mutations located within the central core of the domain severely affect secretion efficiency. In longer α3 chain constructs, spanning N6-N3, small-angle X-ray scattering demonstrates that the tandem VWA array has a modular architecture and samples multiple conformations in solution. Single-particle EM confirmed the presence of multiple conformations. Structural adaptability appears intrinsic to the VWA domain region of collagen VI α3 and has implications for binding interactions and modulating stiffness within the ECM.

The crystal structure of Arabidopsis BON1 provides insights into the copine protein family.

The Arabidopsis thaliana BON1 gene product is a member of the evolutionary conserved eukaryotic calcium-dependent membrane-binding protein family. The copine protein is composed of two C2 domains (C2A and C2B) followed by a vWA domain. The BON1 protein is localized on the plasma membrane, and is known to suppress the expression of immune receptor genes and to positively regulate stomatal closure. The first structure of this protein family has been determined to 2.5-Å resolution and shows the structural features of the three conserved domains C2A, C2B and vWA. The structure reveals the third Ca2+ -binding region in C2A domain is longer than classical C2 domains and a novel Ca2+ binding site in the vWA domain. The structure of BON1 bound to Mn2+ is also presented. The binding of the C2 domains to phospholipid (PSF) has been modeled and provides an insight into the lipid-binding mechanism of the copine proteins. Furthermore, the selectivity of the separate C2A and C2B domains and intact BON1 to bind to different phospholipids has been investigated, and we demonstrated that BON1 could mediate aggregation of liposomes in response to Ca2+ . These studies have formed the basis of further investigations into the important role that the copine proteins play in vivo.

Characterization of a novel shell matrix protein with vWA domain from Mytilus coruscus.

Mollusk shell is a product of biomineralization with excellent mechanical properties, and the shell matrix proteins (SMPs) have important functions in shell formation. A vWA domain-containing protein (VDCP) was identified from the shell of Mytilus coruscus as a novel shell matrix protein. The VDCP gene is expressed at a high level in specific locations in the mantle and adductor muscle. Recombinant VDCP (rVDCP) showed abilities to alter the morphology of both calcite and aragonite, induce the polymorph change of calcite, bind calcite, and decrease the crystallization rate of calcite. In addition, immunohistochemistry analyses revealed the specific location of VDCP in the mantle, the adductor muscle, and the myostracum layer of the shell. Furthermore, a pull-down analysis revealed eight protein interaction partners of VDCP in shell matrices and provided a possible protein-protein interaction network of VDCP in the shell.

A pivotal role for a conserved bulky residue at the α1-helix of the αI integrin domain in ligand binding.

The ligand-binding ßI and αI domains of integrin are the best-studied von Willebrand factor A domains undergoing significant conformational changes for affinity regulation. In both ßI and αI domains, the α1- and α7-helixes work in concert to shift the metal-ion-dependent adhesion site between the resting and active states. An absolutely conserved Gly in the middle of the α1-helix of ßI helps maintain the resting ßI conformation, whereas the homologous position in the αI α1-helix contains a conserved Phe. A functional role of this Phe is structurally unpredictable. Using αLß2 integrin as a model, we found that the residue volume at the Phe position in the α1-helix is critical for αLß2 activation because trimming the Phe by small amino acid substitutions abolished αLß2 binding with soluble and immobilized intercellular cell adhesion molecule 1. Similar results were obtained for αMß2 integrin. Our experimental and molecular dynamics simulation data suggested that the bulky Phe acts as a pawl that stabilizes the downward ratchet-like movement of ß6-α7 loop and α7-helix, required for high-affinity ligand binding. This mechanism may apply to other von Willebrand factor A domains undergoing large conformational changes. We further demonstrated that the conformational cross-talk between αL αI and ß2 ßI could be uncoupled because the ß2 extension and headpiece opening could occur independently of the αI activation. Reciprocally, the αI activation does not inevitably lead to the conformational changes of the ß2 subunit. Such loose linkage between the αI and ßI is attributed to the αI flexibility and could accommodate the αLß2-mediated rolling adhesion of leukocytes.

Heterogeneity of Collagen VI Microfibrils: STRUCTURAL ANALYSIS OF NON-COLLAGENOUS REGIONS.

Collagen VI, a collagen with uncharacteristically large N- and C-terminal non-collagenous regions, forms a distinct microfibrillar network in most connective tissues. It was long considered to consist of three genetically distinct α chains (α1, α2, and α3). Intracellularly, heterotrimeric molecules associate to form dimers and tetramers, which are then secreted and assembled to microfibrils. The identification of three novel long collagen VI α chains, α4, α5, and α6, led to the question if and how these may substitute for the long α3 chain in collagen VI assembly. Here, we studied structural features of the novel long chains and analyzed the assembly of these into tetramers and microfibrils. N- and C-terminal globular regions of collagen VI were recombinantly expressed and studied by small angle x-ray scattering (SAXS). Ab initio models of the N-terminal globular regions of the α4, α5, and α6 chains showed a C-shaped structure similar to that found for the α3 chain. Single particle EM nanostructure of the N-terminal globular region of the α4 chain confirmed the C-shaped structure revealed by SAXS. Immuno-EM of collagen VI extracted from tissue revealed that like the α3 chain the novel long chains assemble to homotetramers that are incorporated into mixed microfibrils. Moreover, SAXS models of the C-terminal globular regions of the α1, α2, α4, and α6 chains were generated. Interestingly, the α1, α2, and α4 C-terminal globular regions dimerize. These self-interactions may play a role in tetramer formation.

A new extracellular von Willebrand A domain-containing protein is involved in silver uptake in Microcystis aeruginosa exposed to silver nanoparticles.

Silver nanoparticles (AgNPs) can be toxic for cyanobacteria when present at low nanomolar concentrations, but the molecular mechanisms whereby AgNPs (or free Ag(+) released from AgNPs) interact with these prokaryotic algal cells remain elusive. Here, we studied Ag uptake mechanisms in the prokaryotic cyanobacterium Microcystis aeruginosa exposed to AgNPs by measuring growth inhibition in the absence or presence of high-affinity Ag-binding ligands and by genetic transformation of E. coli with a protein predicted to be involved in Ag uptake. We discovered a new von Willebrand A (vWA) domain-containing protein in M. aeruginosa that mediates Ag uptake from AgNPs when expressed in E. coli. This new Ag transport protein, which is absent in eukaryotic algae, is a potential candidate explaining the higher AgNPs toxicity in cyanobacteria such as M. aeruginosa than that in eukaryotic algae. The present study provides new insights on Ag uptake mechanisms in the prokaryotic algae M. aeruginosa.

Insights into the tolerant function of VWA proteins in terms of expression analysis and RGLG5-VWA crystal structure.

The VWA domain commonly functions as a crucial component of multiprotein complexes, facilitating protein-protein interactions. However, limited studies have focused on the systemic study of VWA proteins in plants. Here, we identified 28 VWA protein genes in Arabidopsis thaliana, categorized into three clades, with one tandem duplication event and four paralogous genes within collinearity blocks. Then, we determined their expression patterns under abiotic stresses by transcriptomic analysis. All five RGLG genes were found to be responsive to at least one kind of abiotic stress, and RGLG5 was identified as a multiple stress-responsive gene, coding an E3 ubiquitin ligase with a VWA domain and a C-terminal RING domain. Subsequently, we explored tolerant function of RGLG5 by determining the crystal structure of its VWA domain. The structural comparison revealed the allosteric regulation mechanism of RGLG5-VWA, wherein the deflection of α7 led to displacement of key residue binding metal ion within MIDAS motif. Our findings provide full-scale knowledge on VWA proteins, and insights into tolerant function of RGLG5-VWA in terms of crystal structure.

The regulation of RGLG2-VWA by Ca2+ ions.

RGLG2, an E3 ubiquitin ligase in Arabidopsis thaliana, affects hormone signaling and participates in drought regulation. Here, we determined two crystal structures of RGLG2 VWA domain, representing two conformations, open and closed, respectively. The two structures reveal that Ca2+ ions are allosteric regulators of RGLG2-VWA, which adopts open state when NCBS1(Novel Calcium ions Binding Site 1) binds Ca2+ ions and switches to closed state after Ca2+ ions are removed. This mechanism of allosteric regulation is identical to RGLG1-VWA, but distinct from integrin α and ß VWA domains. Therefore, our data provide a backdrop for understanding the role of the Ca2+ ions in conformational change of VWA domain. In addition, we found that RGLG2closed, corresponding to low affinity, can bind pseudo-ligand, which has never been observed in other VWA domains.

Deciphering the enigmatic PilY1 of Acidithiobacillus thiooxidans: An in silico analysis.

Thirty years since the first report on the PilY1 protein in bacteria, only the C-terminal domain has been crystallized; there is no study in which the N-terminal domain, let alone the complete protein, has been crystallized. In our laboratory, we are interested in characterizing the Type IV Pili (T4P) of Acidithiobacillus thiooxidans. We performed an in silico characterization of PilY1 and other pilins of the T4P of this acidophilic bacterium. In silico characterization is crucial for understanding how proteins adapt and function under extreme conditions. By analyzing the primary and secondary structures of proteins through computational methods, researchers can gain valuable insights into protein stability, key structural features, and unique amino acid compositions that contribute to resilience in harsh environments. Here, it is presented a description of the particularities of At. thiooxidans PilY1 through predictor software and homology data. Our results suggest that PilY1 from At. thiooxidans may have the same role as has been described for other PilY1 associated with T4P in neutrophilic bacteria; also, its C-terminal interacts (interface interaction) with the minor pilins PilX, PilW and PilV. The N-terminal region comprises domains such as the vWA and the MIDAS, involved in signaling, ligand-binding, and protein-protein interaction. In fact, the vWA domain has intrinsically disordered regions that enable it to maintain its structure over a wide pH range, not only at extreme acidity to which At. thiooxidans is adapted. The results obtained helped us design the correct methodology for its heterologous expression. This allowed us partially experimentally characterize it by obtaining the N-terminal domain recombinantly and evaluating its acid stability through fluorescence spectroscopy. The data suggest that it remains stable across pH changes. This work thus provides guidance for the characterization of extracellular proteins from extremophilic organisms.

Ca2+-based allosteric switches and shape shifting in RGLG1 VWA domain.

RGLG1 is an E3 ubiquitin ligase in Arabidopsis thaliana that participates in ABA signaling and regulates apical dominance. Here, we present crystal structures of RGLG1 VWA domain, revealing two novel calcium ions binding sites (NCBS1 and NCBS2). Furthermore, the structures with guided mutagenesis in NCBS1 prove that Ca2+ ions play important roles in controlling conformational change of VWA, which is stabilized in open state with Ca2+ bound and converted to closed state after Ca2+ removal. This allosteric regulation mechanism is distinct from the ever reported one involving the C-terminal helix in integrin α and ß I domains. The mutation of a key residue in NCBS2 do not abolish its Ca2+-binding potential, with no conformational change. MD simulations reveals that open state of RGLG1 VWA has higher ligand affinity than its closed state, consisting with integrin. Structural comparison of ion-free-MIDAS with Mg2+-MIDAS reveals that Mg2+ binding to MIDAS does not induce conformational change. With acquisition of first structure of plant VWA domain in both open state and closed state, we carefully analyze the conformational change and propose a totally new paradigm for its transition of open-closed states, which will be of great value for guiding future researches on VWA proteins and their important biological significance.

Matrilins.

Marilins mediate interactions between macromolecular components of the extracellular matrix, e.g., collagens and proteoglycans. They are composed of von Willebrand factor type A and epidermal growth factor-like domains and the subunits oligomerize via coiled-coil domains. Matrilin-1 and -3 are abundant in hyaline cartilage, whereas matrilin-2 and -4 are widespread but less abundant. Mutations in matrilin genes have been linked to chondrodysplasias and osteoarthritis and recently characterization of matrilin-deficient mice revealed novel functions in mechanotransduction, regeneration, or inflammation. Due to their intrinsic adhesiveness and partially also low abundance, the study of matrilins is cumbersome. In this chapter, we describe methods for purification of matrilins from tissue, analysis of matrilins in tissue extracts, recombinant expression, and generation of matrilin-specific antibodies.

Structural Immunology of Complement Receptors 3 and 4.

Complement receptors (CR) 3 and 4 belong to the family of beta-2 (CD18) integrins. CR3 and CR4 are often co-expressed in the myeloid subsets of leukocytes, but they are also found in NK cells and activated T and B lymphocytes. The heterodimeric ectodomain undergoes considerable conformational change in order to switch the receptor from a structurally bent, ligand-binding in-active state into an extended, ligand-binding active state. CR3 binds the C3d fragment of C3 in a way permitting CR2 also to bind concomitantly. This enables a hand-over of complement-opsonized antigens from the cell surface of CR3-expressing macrophages to the CR2-expressing B lymphocytes, in consequence acting as an antigen presentation mechanism. As a more enigmatic part of their functions, both CR3 and CR4 bind several structurally unrelated proteins, engineered peptides, and glycosaminoglycans. No consensus motif in the proteinaceous ligands has been established. Yet, the experimental evidence clearly suggest that the ligands are primarily, if not entirely, recognized by a single site within the receptors, namely the metal-ion dependent adhesion site (MIDAS). Comparison of some recent identified ligands points to CR3 as inclined to bind positively charged species, while CR4, by contrast, binds strongly negative-charged species, in both cases with the critical involvement of deprotonated, acidic groups as ligands for the Mg2+ ion in the MIDAS. These properties place CR3 and CR4 firmly within the realm of modern molecular medicine in several ways. The expression of CR3 and CR4 in NK cells was recently demonstrated to enable complement-dependent cell cytotoxicity toward antibody-coated cancer cells as part of biological therapy, constituting a significant part of the efficacy of such treatment. With the flexible principles of ligand recognition, it is also possible to propose a response of CR3 and CR4 to existing medicines thereby opening a possibility of drug repurposing to influence the function of these receptors. Here, from advances in the structural and cellular immunology of CR3 and CR4, we review insights on their biochemistry and functions in the immune system.