Paul A. McEwan

Quaternary organization of GPIb-IX complex and insights into Bernard-Soulier syndrome revealed by the structures of GPIbβ and a GPIbβ/GPIX chimera

Platelet GPIb-IX receptor complex has 3 subunits GPIbα, GPIbβ, and GPIX, which assemble with a ratio of 1:2:1. Dysfunction in surface expression of the complex leads to Bernard-Soulier syndrome. We have crystallized the GPIbβ ectodomain (GPIbβ(E)) and determined the structure to show a single leucine-rich repeat with N- and C-terminal disulphide-bonded capping regions. The structure of a chimera of GPIbβ(E) and 3 loops (a,b,c) taken from the GPIX ectodomain sequence was also determined. The chimera (GPIbβ(Eabc)), but not GPIbβ(E), forms a tetramer in the crystal, showing a quaternary interface between GPIbβ and GPIX. Central to this interface is residue Tyr106 from GPIbβ, which inserts into a pocket generated by 2 loops (b,c) from GPIX. Mutagenesis studies confirmed this interface as a valid representation of interactions between GPIbβ and GPIX in the full-length complex. Eight GPIbβ missense mutations identified from patients with Bernard-Soulier syndrome were examined for changes to GPIb-IX complex surface expression. Two mutations, A108P and P74R, were found to maintain normal secretion/folding of GPIbβ(E) but were unable to support GPIX surface expression. The close structural proximity of these mutations to Tyr106 and the GPIbβ(E) interface with GPIX indicates they disrupt the quaternary organization of the GPIb-IX complex.

Leucine-rich repeat protein PRAME: expression, potential functions and clinical implications for leukaemia

PRAME/MAPE/OIP4 is a germinal tissue-specific gene that is also expressed at high levels in haematological malignancies and solid tumours. The physiological functions of PRAME in normal and tumour cells are unknown, although a role in the regulation of retinoic acid signalling has been proposed. Sequence homology and structural predictions suggest that PRAME is related to the leucine-rich repeat (LRR) family of proteins, which have diverse functions. Here we review the current knowledge of the structure/function of PRAME and its relevance in leukaemia.

Molecular modeling of the prekallikrein structure provides insights into high-molecular-weight kininogen binding and zymogen activation.

Summary.  Background: Prekallikrein (PK) plays a central role in the contact system that activates blood coagulation and is involved in the regulation of blood pressure.

Glycoprotein Ibalpha inhibitor complex structure reveals a combined steric and allosteric mechanism of von Willebrand factor antagonism.

Platelet glycoprotein Ibalpha (GpIbalpha) interactions with von Willebrand factor (VWF) are a critical early event in platelet adhesion, which contributes to hemostasis and thrombosis. Here we report the structure of a complex between GpIbalpha and a potent peptide inhibitor. The cyclic peptide (CTERMALHNLC) was isolated from a cysteine-constrained phage display library, and in the complex this forms one and a half turns of an amphipathic alpha-helix, the curvature of which facilitates contacts with the curved concave face of the GpIbalpha leucine-rich repeats. The peptide has only limited overlap with the VWF binding site. It effectively inhibits by stabilizing an alternative alpha-helical conformation of a regulatory loop that forms an extended beta-hairpin upon VWF binding. The structure defines a previously unrecognized binding site within GpIbalpha and represents a clear strategy for developing antiplatelet agents targeting the GpIbalpha-VWF interaction allosterically.

Binding of platelet glycoprotein Ibβ through the convex surface of leucine-rich repeats domain of glycoprotein IX

Summary.  Background: The mechanism of assembly of the platelet glycoprotein (GP) Ib‐IX complex from GPIbα, GPIbβ and GPIX subunits is not entirely clear. In this complex, ectodomains of both GPIbβ and GPIX subunits contain two leucine‐rich repeats (LRR) and share high sequence similarity. However, they differ noticeably in stability, hampering further analysis of their interaction. Objectives and methods: Guided by analysis of the LRR structure, we report a well‐folded Ibβ/IX chimera and its usage in dissecting GPIX function. Results: In this chimera, three non‐contiguous sequences that may constitute the putative convex surface of the GPIbβ ectodomain are replaced by their GPIX counterparts. Like GPIbβ but unlike GPIX ectodomain, it can secrete from transfected Chinese hamster ovary cells and fold into a stable conformation. Furthermore, replacing the ectodomain in GPIX with the Ibβ/IX chimera, but not the GPIbβ ectodomain, preserved its interaction with GPIbβ as demonstrated by its native‐like GPIbβ‐induced increase in surface expression and coimmunoprecipitation. Conclusions: The putative convex surface of the LRR domain in GPIX is sufficient, in the context of full‐length subunit, to mediate its association with GPIbβ.

The Structure of Integrin α1I Domain in Complex with a Collagen Mimetic Peptide

Background: Collagen-binding integrins bind differentially to different types of collagen. Results: The solution structure of integrin α1I domain in complex with a collagen-mimetic peptide was determined. Conclusion: Integrin α1I domain binds collagen in a distinct orientation compared with α2I, but the signal transduction mechanisms appear to be conserved. Significance: Understanding the collagen binding specificity of integrins might enable their selective modulation in disease. We have determined the structure of the human integrin α1I domain bound to a triple-helical collagen peptide. The structure of the α1I-peptide complex was investigated using data from NMR, small angle x-ray scattering, and size exclusion chromatography that were used to generate and validate a model of the complex using the data-driven docking program, HADDOCK (High Ambiguity Driven Biomolecular Docking). The structure revealed that the α1I domain undergoes a major conformational change upon binding of the collagen peptide. This involves a large movement in the C-terminal helix of the αI domain that has been suggested to be the mechanism by which signals are propagated in the intact integrin receptor. The structure suggests a basis for the different binding selectivity observed for the α1I and α2I domains. Mutational data identify residues that contribute to the conformational change observed. Furthermore, small angle x-ray scattering data suggest that at low collagen peptide concentrations the complex exists in equilibrium between a 1:1 and 2:1 α1I-peptide complex.

Ring Structure of the Escherichia coli DNA-binding Protein RdgC Associated with Recombination and Replication Fork Repair *

The DNA-binding protein, RdgC, is associated with recombination and replication fork repair in Escherichia coli and with the virulence-associated, pilin antigenic variation mediated by RecA and other recombination proteins in Neisseria species. We solved the structure of the E. coli protein and refined it to 2.4Å. RdgC crystallizes as a dimer with a head-to-head, tail-to-tail organization forming a ring with a 30Å diameter hole at the center. The protein fold is unique and reminiscent of a horseshoe with twin gates closing the open end. The central hole is lined with positively charged residues and provides a highly plausible DNA binding channel consistent with the nonspecific mode of binding detected in vitro and with the ability of RdgC to modulate RecA function in vivo.