José M. Casasnovas

Distinct Kinetics for Binding of the CD46 and SLAM Receptors to Overlapping Sites in the Measles Virus Hemagglutinin Protein

Measles virus (MV) is a human pathogen using two distinct cell surface receptors for entry into host cells. We present here a comparative analysis for binding of the MV receptors CD46 and SLAM to the measles virus hemagglutinin protein (MVH, Edmonston strain). Soluble monomeric and dimeric MVH variants were prepared in mammalian cells and their conformation assessed using a panel of monoclonal antibodies. The two receptor molecules specifically bound to the MVH protein with distinct binding modes. The association rate (k a ) for SLAM binding to MVH was very low (∼3000 m −1s−1), about 20 times lower that the k a determined for CD46 binding. However, SLAM bound tighter to the virus protein than CD46, as revealed by a 5-fold lower dissociation rate (k d , ∼1.5 × 10−3 s−1). These data suggest that the SLAM receptor binds to a less accessible and more hydrophobic surface on MVH than the CD46 receptor, as illustrated in a binding model. Despite the differences in kinetics, receptor competition binding experiments revealed that they recognize overlapping sites in MVH. Indeed, a panel of anti-MVH monoclonal antibodies equally inhibited binding of both receptor molecules. The similar immune reactivity of the two receptor binding sites suggests that the shift in receptor usage by MV may not be driven by immune responses.

Receptor Priming of Major Group Human Rhinoviruses for Uncoating and Entry at Mild Low-pH Environments

ABSTRACT Receptor priming of low-pH-triggered virus entry has been described for an enveloped virus (15). Here we show with major group human rhinoviruses (HRV) and its intercellular adhesion molecule-1 receptor that nonenveloped viruses follow this novel cell entry principle. In vitro the receptor primed HRV for efficient uncoating at mild low pH (5.5 to 6.0). Agents preventing endosomal acidification reduced or blocked rhinovirus cell infection, while nocodazole had no effect on infection of any serotype tested. The entry inhibitory effect of lysosomotropic agents was overcome by exposing cell-internalized HRV to mild low pH (5.5 to 6.0). We therefore conclude that receptor priming of major group HRV must occur in vivo as well. Cooperation of a cellular receptor and low pH in virus uncoating will polarize the exit of the genome to the receptor-bound, membrane-proximal region of the virus particle during acidification of endosomes. This process must be required for efficient penetration of the cellular membrane by viruses.

The structure of immunoglobulin superfamily domains 1 and 2 of MAdCAM-1 reveals novel features important for integrin recognition.

BACKGROUNDnMucosal addressin cell adhesion molecule 1 (MAdCAM-1) is a cell adhesion molecule that is expressed on the endothelium in mucosa, and guides the specific homing of lymphocytes into mucosal tissues. MAdCAM-1 belongs to a subclass of the immunoglobulin superfamily (IgSF), the members of which are ligands for integrins. Human MAdCAM-1 has a unique dual function compared to other members in the same subclass in that it binds both the integrin alpha4beta7, through its two IgSF domains, and a selectin expressed on leukocytes, via carbohydrate sidechains. The structure determination of the two IgSF domains and comparison to the N-terminal two-domain structures of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecules (ICAM-1 and ICAM-2) allow us to assess the molecular basis of the interactions between integrins and their preferred ligands.nnnRESULTSnThe crystal structure of a fragment containing the two IgSF domains of human MAdCAM-1 has been determined to 2.2 A resolution. The structure of MAdCAM-1 reveals two separate integrin-recognition motifs. The key integrin-binding residue, Asp42, resides in the CD loop of domain 1; a buried arginine residue (Arg70) plays a critical role in maintaining the conformation of this loop. The second binding site is associated with an unusual long D strand in domain 2. The D and E strands extend beyond the main body of the domain, forming a negatively charged beta ribbon unique to MAdCAM-1. This ribbon is located on the same face as the key aspartate residue in domain 1, consistent with evidence that it is involved in integrin binding.nnnCONCLUSIONSnThe structural comparison of MAdCAM-1 to other members of the same IgSF subclass reveals some interesting features. Firstly, MAdCAM-1, like VCAM-1, has the key integrin-binding residue located on the protruding CD loop of domain 1 and binds to an integrin that lacks an I domain. This is in contrast to ICAM-1 and ICAM-2 where the key residue is located at the end of the C strand on a flat surface and which bind to integrins that contain I domains. Secondly, architectural differences in the CD loops of MAdCAM-1 and VCAM-1 cause an 8 A shift in position of the critical aspartate residue, and may partly determine their binding preference for different integrins. Finally, the unusual charge distribution of the two-domain fragment of MAdCAM-1 is predicted to orient the molecule optimally for integrin binding on the top of its long mucin-like stalk.

Fructose 1-Phosphate Is the Preferred Effector of the Metabolic Regulator Cra of Pseudomonas putida

The catabolite repressor/activator (Cra) protein is a global sensor and regulator of carbon fluxes through the central metabolic pathways of Gram-negative bacteria. To examine the nature of the effector (or effectors) that signal such fluxes to the protein of Pseudomonas putida, the Cra factor of this soil microorganism has been purified and characterized and its three-dimensional structure determined. Analytical ultracentrifugation, gel filtration, and mobility shift assays showed that the effector-free Cra is a dimer that binds an operator DNA sequence in the promoter region of the fruBKA cluster. Furthermore, fructose 1-phosphate (F1P) was found to most efficiently dissociate the Cra-DNA complex. Thermodynamic parameters of the F1P-Cra-DNA interaction calculated by isothermal titration calorimetry revealed that the factor associates tightly to the DNA sequence 5′-TTAAACGTTTCA-3′ (KD = 26.3 ± 3.1 nm) and that F1P binds the protein with an apparent stoichiometry of 1.06 ± 0.06 molecules per Cra monomer and a KD of 209 ± 20 nm. Other possible effectors, like fructose 1,6-bisphosphate, did not display a significant affinity for the regulator under the assay conditions. Moreover, the structure of Cra and its co-crystal with F1P at a 2-Å resolution revealed that F1P fits optimally the geometry of the effector pocket. Our results thus single out F1P as the preferred metabolic effector of the Cra protein of P. putida.

Allosteric inhibition of aminopeptidase N functions related to tumor growth and virus infection

Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme.

Crystallization and preliminary crystallographic analysis of the measles virus hemagglutinin in complex with the CD46 receptor

The measles virus (MV) hemagglutinin (MV-H) mediates the attachment of MV particles to cell-surface receptors for entry into host cells. MV uses two receptors for attachment to host cells: the complement-control protein CD46 and the signalling lymphocyte activation molecule (SLAM). The MV-H glycoprotein from an Edmonston MV variant and the MV-binding fragment of the CD46 receptor were overproduced in mammalian cells and used to crystallize an MV-H-CD46 complex. Well diffracting crystals containing two complexes in the asymmetric unit were obtained and the structure of the complex was solved by the molecular-replacement method.