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Dusan Uhrin

Publications and source records attributed to Dusan Uhrin.

7 recordsLinked to original sources
PubMed2005

Human C4b-binding protein, structural basis for interaction with streptococcal M protein, a major bacterial virulence factor.

Human C4b-binding protein (C4BP) protects host tissue, and those pathogens able to hijack this plasma glycoprotein, from complement-mediated destruction. We now show that the first two complement control protein (CCP) modules of the C4BP alpha-chain, plus the four residues connecting them, are necessary and sufficient for binding a bacterial virulence factor, the Streptococcus pyogenes M4 (Arp4) protein. Structure determination by NMR reveals two tightly coupled CCP modules in an elongated arrangement within this region of C4BP. Chemical shift perturbation studies demonstrate that the N-terminal, hypervariable region of M4 binds to a site including strand 1 of CCP module 2. This interaction is accompanied by an intermodular reorientation within C4BP. We thus provide a detailed picture of an interaction whereby a pathogen evades complement.

Amino Acid Sequence↗
PubMed2005

Structure of free MDM2 N-terminal domain reveals conformational adjustments that accompany p53-binding.

Critical to the inhibitory action of the oncogene product, MDM2, on the tumour suppressor, p53, is association of the N-terminal domain of MDM2 (MDM2N) with the transactivation domain of p53. The structure of MDM2N was previously solved with a p53-derived peptide, or small-molecule ligands, occupying its binding cleft, but no structure of the non-liganded MDM2N (i.e. the apo-form) has been reported. Here, we describe the solution structure and dynamics of apo-MDM2N and thus reveal the nature of the conformational changes in MDM2N that accompany binding of p53. The new structure suggests that p53 effects displacement of an N-terminal segment of apo-MDM2N that occludes access to the shallow end of the p53-binding cleft. MDM2N must also undergo an expansion upon binding, achieved through a rearrangement of its two pseudosymetrically related sub-domains resulting in outward displacements of the secondary structural elements that comprise the walls and floor of the p53-binding cleft. MDM2N becomes more rigid and stable upon binding p53. Conformational plasticity of the binding cleft of apo-MDM2N could allow the parent protein to bind specifically to several different partners, although, to date, all the known liganded structures of MDM2N are highly similar to one another. The results indicate that the more open conformation of the binding cleft of MDM2N observed in structures of complexes with small molecules and peptides is a more suitable one for ligand discovery and optimisation.

Animals↗
PubMed2004

Structural analysis of the complement control protein (CCP) modules of GABA(B) receptor 1a: only one of the two CCP modules is compactly folded.

The gamma-aminobutyric acid type B (GABA(B)) receptor is a heterodimeric G-protein-coupled receptor. In humans, three splice variants of the GABA(B) receptor 1 (R1) subunit differ in having one, both, or neither of two putative complement control protein (CCP) modules at the extracellular N terminus, prior to the GABA-binding domain. The in vivo function of these predicted modules remains to be discovered, but a likely association with extracellular matrix proteins is intriguing. The portion of the GABA(B) R1a variant encompassing both of its CCP module-like sequences has been expressed, as have the sequences corresponding to each individual module. Each putative CCP module exhibits the expected pattern of disulfide formation. However, the second module (CCP2) is more compactly folded than the first, and the three-dimensional structure of this more C-terminal module (expressed alone) was solved on the basis of NMR-derived nuclear Overhauser effects. This revealed a strong similarity to previously determined CCP module structures in the regulators of complement activation. The N-terminal module (CCP1) displayed conformational heterogeneity under a wide range of conditions whether expressed alone or together with CCP2. Several lines of evidence indicated the presence of native disorder in CCP1, despite the fact that recombinant CCP1 contributes to binding to the extracellular matrix protein fibulin-2. Thus, we have shown that the two CCP modules of GABA(B) R1a have strikingly different structural properties, reflecting their different functions.

Animals↗
PubMed2004

Backbone dynamics of complement control protein (CCP) modules reveals mobility in binding surfaces.

The regulators of complement activation (RCA) are critical to health and disease because their role is to ensure that a complement-mediated immune response to infection is proportionate and targeted. Each protein contains an uninterrupted array of from four to 30 examples of the very widely occurring complement control protein (CCP, or sushi) module. The CCP modules mediate specific protein-protein and protein-carbohydrate interactions that are key to the biological function of the RCA and, paradoxically, provide binding sites for numerous pathogens. Although structural and mutagenesis studies of CCP modules have addressed some aspects of molecular recognition, there have been no studies of the role of molecular dynamics in the interaction of CCP modules with their binding partners. NMR has now been used in the first full characterization of the backbone dynamics of CCP modules. The dynamics of two individual modules-the 16th of the 30 modules of complement receptor type 1 (CD35), and the N-terminal module of membrane cofactor protein (CD46)-as well as their solution structures, are compared. Although both examples share broadly similar three-dimensional structures, many structurally equivalent residues exhibit different amplitudes and timescales of local backbone motion. In each case, however, regions of the module-surface implicated by mutagenesis as sites of interactions with other proteins include several mobile residues. This observation suggests further experiments to explore binding mechanisms and identify new binding sites.

Amino Acid Sequence↗
PubMed2003

Solution structure of a functionally active fragment of decay-accelerating factor.

The second and third modules of human decay accelerating factor (DAF) are necessary and sufficient to accelerate decay of the classical pathway (CP) convertase of complement. No structure of a mammalian protein with decay-accelerating activity has been available to date. We therefore determined the solution structure of DAF modules 2 and 3 (DAF approximately 2,3). Structure-guided analysis of 24 mutants identified likely contact points between DAF and the CP convertase. Three (R96, R69, and a residue in the vicinity of L171) lie on DAF approximately 2,3's concave face. A fourth, consisting of K127 and nearby R100, is on the opposite face. Regions of module 3 remote from the semiflexible 2-3 interface seem not to be involved in binding to the CP convertase. DAF thus seems to occupy a groove on the CP convertase such that both faces of DAF close to the 2-3 junction (including a positively charged region that encircles the protein at this point) interact simultaneously. Alternative pathway convertase interactions with DAF require additional regions of CCP 3 lying away from the 2-3 interface, consistent with the established additional requirement of module 4 for alternative pathway regulation.

Binding Sites↗
PubMed2002

Structure of the C3b binding site of CR1 (CD35), the immune adherence receptor.

Complement receptor type 1 (CR1 or CD35) is a multiple modular protein that mediates the immune adherence phenomenon, a fundamental event for destroying microbes and initiating an immunological response. It fulfills this role through binding C3b/C4b-opsonized foreign antigens. The structure of the principal C3b/C4b binding site (residues 901-1095) of CR1 is reported, revealing three complement control protein modules (modules 15-17) in an extended head-to-tail arrangement with flexibility at the 16-17 junction. Structure-guided mutagenesis identified a positively charged surface region on module 15 that is critical for C4b binding. This patch, together with basic side chains of module 16 exposed on the same face of CR1, is required for C3b binding. These studies reveal the initial structural details of one of the first receptor-ligand interactions to be identified in immunobiology.

Binding Sites↗