Manuela Bozzi

Plasticity of secondary structure in the N-terminal region of β-dystroglycan

The secondary structure content of the N-terminal extracellular domain of beta-dystroglycan (a recombinant fragment extending from positions 654 to 750) has been quantitatively determined by means of CD and FTIR spectroscopies. The elements of secondary structure, namely an 8-10 residue long alpha-helix (10%) and two beta-strands (24%) have been assigned to specific amino acid sequences by means of a GOR constrained prediction method. The remaining 66% of the whole sequence is classified as turns or unordered. The temperature dependence of CD and FTIR spectra has been investigated in detail. A reversible, non-cooperative thermal transition is observed with both CD and FTIR spectroscopies up to 95 degrees C. The profile of the transition is typical of the unfolding of isolated peptides and corresponds to the progressive loss of the secondary structure elements of the protein with no evidence for collapsing phenomena, typical of globular proteins, upon heating.

Spectroscopic and molecular dynamics simulation studies of the interaction of insulin with glucose

The interaction between monomeric insulin and monosaccharides has been investigated through circular dichroism, fluorescence spectroscopy and two dimensional nuclear magnetic resonance. CD spectra indicate that D-glucose interacts with monomeric insulin whereas D-galactose, D-mannose and 2-deoxy-D-glucose have a lower effect. Fluorescence emission was quenched at sugar concentrations of 5-10 mM. Titration with the different sugars produces a quenching of the tyrosine spectrum from which a binding free energy value for the insulin-sugar complexes has been evaluated. Transfer nuclear Overhauser enhancement NMR experiments indicate the existence of dipolar interactions at short interatomic distances between C-1 proton of D-glucose in the beta form and the monomeric insulin. Further, NMR total correlation spectra experiments revealed that the hormone is in the monomeric form and that upon addition of glucose no aggregation occurs. The interaction does not involve relevant changes in the secondary structure of insulin suggesting that the interaction occur at the side chain level. Molecular dynamics simulations and modeling studies, based on the dynamic fluctuations of potential binding moiety sidechains, argued from results of NMR spectroscopy, provide additional informations to locate the putative binding sites of D-glucose to insulin.

A second Ig-like domain identified in dystroglycan by molecular modelling and dynamics

Dystroglycan (DG) is a cell surface receptor which is composed of two subunits that interact noncovalently, namely α- and β-DG. In skeletal muscle, DG is the central component of the dystrophin-glycoprotein complex (DGC) that anchors the actin cytoskeleton to the extracellular matrix. To date only the three-dimensional structure of the N-terminal region of α-DG has been solved by X-ray crystallography. To expand such a structural analysis, a theoretical molecular model of the murine α-DG C-terminal region was built based on folding recognition/threading techniques. Although there is no a significant (<30%) sequence homology with the N-terminal region of α-DG, protein fold recognition methods found a significant resemblance to the α-DG N-terminal crystallographic structure. Our in silico structural prediction identified two subdomains in this region. Amino acid residues ∼ 500-600 of α-DG were predicted to adopt an immunoglobulin-like (Ig-like) β-sandwich fold. Such modeled domain includes the β-DG binding epitope of α-DG and, confirming our previous experimental results, suggests that the linear epitope (residues 550-565) assumes a β-strand conformation. The remaining segment of the α-DG C-terminal region (residues 601-653) is organized in a coil-helix-coil motif. A 20-ns molecular dynamics simulation in explicit water solvent provided support to the predicted Ig-like model structure. The identification of a second Ig-like domain in DG represents another important step towards a full structural and functional description of the α/β DG interface. Preliminary characterization of a novel recombinant peptide (505-600) encompassing this second Ig-like domain demonstrates that it is soluble and stable, further corroborating our in silico analysis.

Enzymatic processing of beta-dystroglycan recombinant ectodomain by MMP-9: identification of the main cleavage site.

Dystroglycan (DG) is a membrane receptor belonging to the complex of glycoproteins associated to dystrophin. DG is formed by two subunits, α‐DG, a highly glycosylated extracellular matrix protein, and β‐DG, a transmembrane protein. The two DG subunits interact through the C‐terminal domain of α‐DG and the N‐terminal extracellular domain of β‐DG in a noncovalent way. Such interaction is crucial to maintain the integrity of the plasma membrane. In some pathological conditions, the interaction between the two DG subunits may be disrupted by the proteolytic activity of gelatinases (i.e. MMP‐9 and/or MMP‐2) that removes a portion or the whole β‐DG ectodomain producing a 30 kDa truncated form of β‐DG. However, the molecular mechanism underlying this event is still unknown. In this study, we carried out proteolysis of the recombinant extracellular domain of β‐DG, β‐DG(654‐750) with human MMP‐9, characterizing the catalytic parameters of its cleavage. Furthermore, using a combined approach based on SDS‐PAGE, MALDI‐TOF and HPLC‐ESI‐IT mass spectrometry, we were able to identify one main MMP‐9 cleavage site that is localized between the amino acids His‐715 and Leu‐716 of β‐DG, and we analysed the proteolytic fragments of β‐DG(654‐750) produced by MMP‐9 enzymatic activity.

Enzymatic processing by MMP-2 and MMP-9 of wild-type and mutated mouse β-dystroglycan

Dystroglycan (DG) is a membrane‐associated protein complex formed by two noncovalently linked subunits, α‐DG, a highly glycosylated extracellular protein, and β‐DG, a transmembrane protein. The interface between the two DG subunits, which is crucial to maintain the integrity of the plasma membrane, involves the C‐terminal domain of α‐DG and the N‐terminal extracellular domain of β‐DG. It is well known that under both, physiological and pathological conditions, gelatinases (i.e. MMP‐9 and/or MMP‐2) can degrade DG, disrupting the connection between the extracellular matrix and the cytoskeleton. However, the molecular mechanisms and the exact cleavage sites underlying these events are still largely unknown. In a previous study, we have characterized the enzymatic digestion of the murine β‐DG ectodomain by gelatinases, identifying a main cleavage site on the β‐DG ectodomain produced by MMP‐9. In this article, we have deepened the pattern of the β‐DG ectodomain digestion by MMP‐2 by using a combined approach based on SDS‐PAGE, Orbitrap, and HPLC‐ESI‐IT mass spectrometry. Furthermore, we have characterized the kineticparameters of the digestion of some β‐DG ectodomain mutants by gelatinases.

Insertion of a myc-tag within α-dystroglycan domains improves its biochemical and microscopic detection

BackgroundEpitope tags and fluorescent fusion proteins have become indispensable molecular tools for studies in the fields of biochemistry and cell biology. The knowledge collected on the subdomain organization of the two subunits of the adhesion complex dystroglycan (DG) enabled us to insert the 10 amino acids myc-tag at different locations along the α-subunit, in order to better visualize and investigate the DG complex in eukaryotic cells.ResultsWe have generated two forms of DG polypeptides via the insertion of the myc-tag 1) within a flexible loop (between a.a. 170 and 171) that separates two autonomous subdomains, and 2) within the C-terminal domain in position 500. Their analysis showed that double-tagging (the β-subunit is linked to GFP) does not significantly interfere with the correct processing of the DG precursor (pre-DG) and confirmed that the α-DG N-terminal domain is processed in the cell before α-DG reaches its plasma membrane localization. In addition, myc insertion in position 500, right before the second Ig-like domain of α-DG, proved to be an efficient tool for the detection and pulling-down of glycosylated α-DG molecules targeted at the membrane.ConclusionsFurther characterization of these and other myc-permissive site(s) will represent a valid support for the study of the maturation process of pre-DG and could result in the creation of a new class of intrinsic doubly-fluorescent DG molecules that would allow the monitoring of the two DG subunits, or of pre-DG, in cells without the need of antibodies.

Mutagenesis at the α–β interface impairs the cleavage of the dystroglycan precursor

The interaction between α‐dystroglycan (α‐DG) and β‐dystroglycan (β‐DG), the two constituent subunits of the adhesion complex dystroglycan, is crucial in maintaining the integrity of the dystrophin–glycoprotein complex. The importance of the α–β interface can be seen in the skeletal muscle of humans affected by severe conditions, such as Duchenne muscular dystrophy, where the α–β interaction can be secondarily weakened or completely lost, causing sarcolemmal instability and muscular necrosis. The reciprocal binding epitopes of the two subunits reside within the C‐terminus of α‐DG and the ectodomain of β‐DG. As no ultimate structural data are yet available on the α–β interface, site‐directed mutagenesis was used to identify which specific amino acids are involved in the interaction. A previous alanine‐scanning analysis of the recombinant β‐DG ectodomain allowed the identification of two phenylalanines important for α‐DG binding, namely F692 and F718. In this article, similar experiments performed on the α‐DG C‐terminal domain pinpointed two residues, G563 and P565, as possible binding counterparts of the two β‐DG phenylalanines. In 293‐Ebna cells, the introduction of alanine residues instead of F692, F718, G563 and P565 prevented the cleavage of the DG precursor that liberates α‐ and β‐DG, generating a pre‐DG of about 160 kDa. This uncleaved pre‐DG tetramutant is properly targeted at the cell membrane, is partially glycosylated and still binds laminin in pull‐down assays. These data reinforce the notion that DG processing and its membrane targeting are two independent processes, and shed new light on the molecular mechanism that drives the maturation of the DG precursor.

Role of gelatinases in pathological and physiological processes involving the dystrophin-glycoprotein complex

Dystrophin is a cytosolic protein belonging to a membrane-spanning glycoprotein complex, called dystrophin-glycoprotein complex (DGC) that is expressed in many tissues, especially in skeletal muscle and in the nervous system. The DGC connects the cytoskeleton to the extracellular matrix and, although none of the proteins of the DGC displays kinase or phosphatase activity, it is involved in many signal transduction pathways. Mutations in some components of the DGC are linked to many forms of inherited muscular dystrophies. In particular, a mutation in the dystrophin gene, leading to a complete loss of the protein, provokes one of the most prominent muscular dystrophies, the Duchenne muscular dystrophy, which affects 1 out of 3500 newborn males. What is observed in these circumstances, is a dramatic alteration of the expression levels of a multitude of metalloproteinases (MMPs), a family of extracellular Zn(2+)-dependent endopeptidases, in particular of MMP-2 and MMP-9, also called gelatinases. Indeed, the enzymatic activity of MMP-2 and MMP-9 on dystroglycan, an important member of the DGC, plays a significant role also in physiological processes taking place in the central and peripheral nervous system. This mini-review discusses the role of MMP-2 and MMP-9, in physiological as well as pathological processes involving members of the DGC.

The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy

The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis in vitro and in vivo indicated that the mutation was responsible for a decrease in posttranslational glycosylation of dystroglycan, eventually interfering with its extracellular-matrix receptor function and laminin binding in skeletal muscle and brain. The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology (Ig-like domain followed by a basket-shaped domain reminiscent of the small subunit ribosomal protein S6) very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159–180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1–B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.

Insights from molecular dynamics simulations: structural basis for the V567D mutation-induced instability of zebrafish alpha-dystroglycan and comparison with the murine model.

A missense amino acid mutation of valine to aspartic acid in 567 position of alpha-dystroglycan (DG), identified in dag1-mutated zebrafish, results in a reduced transcription and a complete absence of the protein. Lacking experimental structural data for zebrafish DG domains, the detailed mechanism for the observed mutation-induced destabilization of the DG complex and membrane damage, remained unclear. With the aim to contribute to a better clarification of the structure-function relationships featuring the DG complex, three-dimensional structural models of wild-type and mutant (V567D) C-terminal domain of alpha-DG from zebrafish were constructed by a template-based modelling approach. We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability. A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish. Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein. A biochemical analysis of the murine alpha-DG mutant I591D confirmed a pronounced instability of the protein. Taken together, the computational and biochemical analysis suggest that the V567D/I591D mutation, belonging to the G beta-strand, plays a key role in inducing a destabilization of the alpha-DG C-terminal Ig-like domain that could possibly affect and propagate to the entire DG complex. The structural features herein identified may be of crucial help to understand the molecular basis of primary dystroglycanopathies.