Jonas Emsley

Structural Basis of Collagen Recognition by Integrin α2β1

We have determined the crystal structure of a complex between the I domain of integrin alpha2beta1 and a triple helical collagen peptide containing a critical GFOGER motif. Three loops on the upper surface of the I domain that coordinate a metal ion also engage the collagen, with a collagen glutamate completing the coordination sphere of the metal. Comparison with the unliganded I domain reveals a change in metal coordination linked to a reorganization of the upper surface that together create a complementary surface for binding collagen. Conformational changes propagate from the upper surface to the opposite pole of the domain, suggesting both a basis for affinity regulation and a pathway for signal transduction. The structural features observed here may represent a general mechanism for integrin-ligand recognition.

A Common Fold Mediates Vertebrate Defense and Bacterial Attack

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.

Crystal structure of the platelet glycoprotein Ib(alpha) N-terminal domain reveals an unmasking mechanism for receptor activation.

Glycoprotein Ib (GPIb) is a platelet receptor with a critical role in mediating the arrest of platelets at sites of vascular damage. GPIb binds to the A1 domain of von Willebrand factor (vWF-A1) at high blood shear, initiating platelet adhesion and contributing to the formation of a thrombus. To investigate the molecular basis of GPIb regulation and ligand binding, we have determined the structure of the N-terminal domain of the GPIbα chain (residues 1–279). This structure is the first determined from the cell adhesion/signaling class of leucine-rich repeat (LRR) proteins and reveals the topology of the characteristic disulfide-bonded flanking regions. The fold consists of an N-terminal β-hairpin, eight leucine-rich repeats, a disulfide-bonded loop, and a C-terminal anionic region. The structure also demonstrates a novel LRR motif in the form of an M-shaped arrangement of three tandem β-turns. Negatively charged binding surfaces on the LRR concave face and anionic region indicate two-step binding kinetics to vWF-A1, which can be regulated by an unmasking mechanism involving conformational change of a key loop. Using molecular docking of the GPIb and vWF-A1 crystal structures, we were also able to model the GPIb·vWF-A1 complex.

Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle.

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin‐mediated cell adhesion and migration. We have determined the crystal structures of two domains from the talin rod spanning residues 482–789. Talin 482–655, which contains a vinculin‐binding site (VBS), folds into a five‐helix bundle whereas talin 656–789 is a four‐helix bundle. We show that the VBS is composed of a hydrophobic surface spanning five turns of helix 4. All the key side chains from the VBS are buried and contribute to the hydrophobic core of the talin 482–655 fold. We demonstrate that the talin 482–655 five‐helix bundle represents an inactive conformation, and mutations that disrupt the hydrophobic core or deletion of helix 5 are required to induce an active conformation in which the VBS is exposed. We also report the crystal structure of the N‐terminal vinculin head domain in complex with an activated form of talin. Activation of the VBS in talin and the recruitment of vinculin may support the maturation of small integrin/talin complexes into more stable adhesions.

Mapping and consensus sequence identification for multiple vinculin binding sites within the talin rod

The interaction between the cytoskeletal proteins talin and vinculin plays a key role in integrin-mediated cell adhesion and migration. Three vinculin binding sites (VBS1-3) have previously been identified in the talin rod using a yeast two-hybrid assay. To extend these studies, we spot-synthesized a series of peptides spanning all the α-helical regions predicted for the talin rod and identified eight additional VBSs, two of which overlap key functional regions of the rod, including the integrin binding site and C-terminal actin binding site. The talin VBS α-helices bind to a hydrophobic cleft in the N-terminal vinculin Vd1 domain. We have defined the specificity of this interaction by spot-synthesizing a series of 25-mer talin VBS1 peptides containing substitutions with all the commonly occurring amino acids. The consensus for recognition is LXXAAXXVAXX- VXXLIXXA with distinct classes of hydrophobic side chains at positions 1, 4, 5, 8, 9, 12, 15, and 16 required for vinculin binding. Positions 1, 8, 12, 15, and 16 require an aliphatic residue and will not tolerate alanine, whereas positions 4, 5, and 9 are less restrictive. These preferences are common to all 11 VBS sequences with a minor variation occurring in one case. A crystal structure of this variant VBS peptide in complex with the vinculin Vd1 domain reveals a subtly different mode of vinculin binding.

FXa variants advance toward a therapy for bleeding.

In this issue of Blood , [Ivanciu and Camire][1] describe engineered factor (F)Xa variants with a spectrum of properties that broaden the utility of FXa as a treatment for bleeding.[1][2] ![Figure][3] Representation of the FXa residues Val17 and Ile16 (purple) buried in the FXa active site

Integrin connections to the cytoskeleton through talin and vinculin

Integrins are alphabeta heterodimeric receptors that mediate attachment of cells to the extracellular matrix and therefore play important roles in cell adhesion, migration, proliferation and survival. Among the cytoskeletal proteins that interact directly with the beta-chain cytoplasmic domain, talin has emerged as playing a critical role in integrin activation and linkage to the actin cytoskeleton. Talin (2541 amino acids) is an elongated (60 nm) flexible antiparallel dimer, with a small globular head connected to an extended rod. The talin head contains a FERM (4.1/ezrin/radixin/moesin) domain (residues 86-400) with binding sites for several beta integrin cytodomains and the talin rod contains a second lower-affinity integrin-binding site, a highly conserved C-terminal actin-binding site and also several binding sites for vinculin. We have determined previously the crystal structures of two domains from the talin rod, spanning residues 482-789. Talin-(482-655), which contains a VBS (vinculin-binding site), folds into a five-helix bundle whereas talin-(656-789) is a four-helix bundle. We have also reported the crystal structure of the N-terminal vinculin head domain in complex with an activated form of talin. In the present paper, we consider how binding sites buried within the folded helical bundles of talin and alpha-actinin form interactions with vinculin.

Staphylococcus aureus protein A binding to von Willebrand factor A1 domain is mediated by conserved IgG binding regions

Protein A (Spa) is a surface‐associated protein of Staphylococcus aureus best known for its ability to bind to the Fc region of IgG. Spa also binds strongly to the Fab region of the immunoglobulins bearing VH3 heavy chains and to von Willebrand factor (vWF). Previous studies have suggested that the protein A–vWF interaction is important in S. aureus adherence to platelets under conditions of shear stress. We demonstrate that Spa expression is sufficient for adherence of bacteria to immobilized vWF under low fluid shear. The full length recombinant Ig‐binding region of protein A, Spa‐EDABC, fused to glutathione‐S‐transferase (GST), bound recombinant vWF in a dose‐dependent and saturable fashion with half maximal binding of about 30 nm in immunosorbent assays. Full length‐Spa did not bind recombinant vWF A3 domain but displayed binding to recombinant vWF domains A1 and D′‐D3 (half maximal binding at 100 nm and 250 nm, respectively). Each recombinant protein A Ig‐binding domain bound to the A1 domain in a similar manner to the full length‐Spa molecule (half maximal binding 100 nm). Amino acid substitutions were introduced in the GST‐SpaD protein at sites known to be involved in IgG Fc or in VH3 Fab binding. Mutants altered in residues that recognized IgG Fc but not those that recognized VH3 Fab had reduced binding to vWF A1 and D′‐D3. This indicated that both vWF regions recognized a region on helices I and II that overlapped the IgG Fc binding site.

Structural Basis for Native Agonist and Synthetic Inhibitor Recognition by the Pseudomonas aeruginosa Quorum Sensing Regulator PqsR (MvfR)

Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4-hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH2) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR.

Structural Determinants of Integrin Binding to the Talin Rod

The adaptor protein talin serves both to activate the integrin family of cell adhesion molecules and to couple integrins to the actin cytoskeleton. Integrin activation has been shown to involve binding of the talin FERM domain to membrane proximal sequences in the cytoplasmic domain of the integrin β-subunit. However, a second integrin-binding site (IBS2) has been identified near the C-terminal end of the talin rod. Here we report the crystal structure of IBS2 (residues 1974-2293), which comprises two five-helix bundles, “IBS2-A” (1974-2139) and “IBS2-B” (2140-2293), connected by a continuous helix with a distinct kink at its center that is stabilized by side-chain H-bonding. Solution studies using small angle x-ray scattering and NMR point to a fairly flexible quaternary organization. Using pull-down and enzyme-linked immunosorbent assays, we demonstrate that integrin binding requires both IBS2 domains, as does binding to acidic phospholipids and robust targeting to focal adhesions. We have defined the membrane proximal region of the integrin cytoplasmic domain as the major binding region, although more membrane distal regions are also required for strong binding. Alanine-scanning mutagenesis points to an important electrostatic component to binding. Thermal unfolding experiments show that integrin binding induces conformational changes in the IBS2 module, which we speculate are linked to vinculin and membrane binding.