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Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to "evade" the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X‐ray crystallography. Coating a protein with PEG may have structural consequences, but no single biophysical methodology can spot the possible modifications. This limitation has been overcome by the integration of solution and solid‐state NMR data, relaxometry, and X‐ray crystallography for the characterization of a PEGylated enzyme used in clinical practice. [ABSTRACT FROM AUTHOR]

The Escherichia coli gene aphA codes for a periplasmic acid phosphatase called AphA, belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases. After our first report about its crystal structure, we have started a series of crystallographic studies aimed at understanding of the catalytic mechanism of the enzyme. Here, we report three crystal structures of the AphA enzyme in complex with the hydrolysis products of nucleoside monophosphate substrates and a fourth with a proposed intermediate analogue that appears to be covalently bound to the enzyme. Comparison with the native enzyme structure and with the available X-ray structures of different phosphatases provides clues about the enzyme chemistry and allows us to propose a catalytic mechanism for AphA, and to discuss it with respect to the mechanism of other bacterial and human phosphatases. [Copyright &y& Elsevier]

The targeted delivery of non-polar ligands by binding proteins to membranes or membrane receptors involves the release of these ligands on or near the plasma membrane of target cells. Because these hydrophobic ligands are often bound inside a deep cavity of binding proteins, as shown previously for plasma retinol-binding protein (RBP), their release from these proteins might require the destabilization of the protein structure by partially denaturing conditions, such as those possibly present near plasma membranes. RBP is a plasma transport protein which delivers specifically retinol from its store sites to target cells. Here, we report the high-resolution (1.1–1.4 A˚) crystal structures of bovine holo-RBP at five different pH values, ranging from 9 to 2. While unraveling details of the native protein structure and of the interactions with retinol at nearly atomic resolution at neutral pH, this study provides evidence for definite pH-induced modifications of several structural features of RBP. The structure most representative of the changes that holo-RBP undergoes at different pH values is that of its flexible state at pH 2. At this pH, most significant are the alteration of the arrangement of salt bridges and of the network of water molecules/H-bonds that participates in the retinol–RBP interaction, an appreciable increase of the volume of the β-barrel cavity, a considerably higher degree of mobility of the RBP-bound ligand and of several protein regions and the disorder of a large number of solvent molecules that are ordered at neutral pH. These changes are likely to be accompanied by a modification of the pattern of charge distribution on the protein surface. All these changes, which reveal a substantially lowered conformational stability of RBP, presumably occur at the initial stages of the acidic denaturation of RBP and are possibly associated with a facilitated release of the retinol molecule from its carrier protein. [Copyright &y& Elsevier]

The molecular determinants responsible for the potencyof the RXP470.1phosphinic peptide inhibitor toward matrix metalloprotease-12 (MMP-12)remain elusive. To address this issue, structure–activity study,X-ray crystallography, and isothermal titration calorimetry (ITC)experiments were performed. The crystal structure of MMP-12/inhibitorcomplex (1.15 Å) reveals that the inhibitor establishes multipleinteractions with the MMP-12 active site, with its long P1′ side chain filling most of the S1′ deepcavity. ITC experiments indicate that the binding of this inhibitorto MMP-12 is mostly entropy driven (ΔG°= −13.1 kcal/mol, ΔH° = −2.53kcal/mol, and −TΔS°= −10.60 kcal/mol) and involves a proton uptake from the buffer.Comparing phosphinic versus hydroxamate inhibitors reveals that thechelation of the zinc ion is slightly different, leading the inhibitorbackbone to adopt a position in which the hydrogen bonding with theMMP-12 active site is less favorable in phosphinic inhibitor whilemaintaining high affinity. [ABSTRACT FROM AUTHOR]

Copper-containing amine oxidase extracted from bovine serum (BSAO) was crystallized and its three-dimensional structure at 2.37Å resolution is described. The biological unit of BSAO is a homodimer, formed by two monomers related to each other by a non-crystallographic 2-fold axis. Each monomer is composed of three domains, similar to those of other amine oxidases from lower species. The two monomers are structurally equivalent, despite some minor differences at the two active sites. A large funnel allows access of substrates to the active-site; another cavity, accessible to the solvent, is also present between the two monomers; this second cavity could allow the entrance of molecular oxygen necessary for the oxidative reaction. Some sugar residues, bound to Asn, were still present and visible in the electron density map, in spite of the exhaustive deglycosylation necessary to grow the crystals. The comparison of the BSAO structure with those of other resolved AO structures shows strong dissimilarities in the architecture and charge distribution of the cavities leading to the active-site, possibly explaining the differences in substrate specificity. [Copyright &y& Elsevier]