Vito Calderone

Crystal Structure of the OXA-48 β-Lactamase Reveals Mechanistic Diversity among Class D Carbapenemases

Carbapenem-hydrolyzing class D beta-lactamases (CHDLs) are enzymes found in important Gram-negative pathogens (mainly Acinetobacter baumannii and Enterobacteriaceae) that confer resistance to beta-lactam antibiotics, and notably carbapenems. The crystal structure of the OXA-48 carbapenemase was determined at pH 7.5 and at a resolution of 1.9 A. Surprisingly, and by contrast with OXA-24, the only other CHDL of known crystal structure, the structure of OXA-48 was similar to OXA-10, an enzyme devoid of carbapenemase activity, indicating that the hydrolysis of these compounds could depend on subtle changes in the active site region. Moreover, the active site groove of OXA-48 was different from that of OXA-24 in shape, dimensions, and charge distribution. Molecular dynamics pointed to the functional relevance of residues located in or close to the beta5-beta6 loop and allowed us to propose a mechanism for carbapenem hydrolysis by OXA-48.

Structural and dynamic aspects related to oligomerization of apo SOD1 and its mutants

The structural and dynamical properties of the metal-free form of WT human superoxide dismutase 1 (SOD1) and its familial amyotrophic lateral sclerosis (fALS)-related mutants, T54R and I113T, were characterized both in solution, through NMR, and in the crystal, through X-ray diffraction. We found that all 3 X-ray structures show significant structural disorder in 2 loop regions that are, at variance, well defined in the fully-metalated structures. Interestingly, the apo state crystallizes only at low temperatures, whereas all 3 proteins in the metalated form crystallize at any temperature, suggesting that crystallization selects one of the most stable conformations among the manifold adopted by the apo form in solution. Indeed, NMR experiments show that the protein in solution is highly disordered, sampling a large range of conformations. The large conformational variability of the apo state allows the free reduced cysteine Cys-6 to become highly solvent accessible in solution, whereas it is essentially buried in the metalated state and the crystal structures. Such solvent accessibility, together with that of Cys-111, accounts for the tendency to oligomerization of the metal-free state. The present results suggest that the investigation of the solution state coupled with that of the crystal state can provide major insights into SOD1 pathway toward oligomerization in relation to fALS.

Evidence of reciprocal reorientation of the catalytic and hemopexin-like domains of full-length MMP-12

The proteolytic activity of matrix metalloproteinases toward extracellular matrix components (ECM), cytokines, chemokines, and membrane receptors is crucial for several homeostatic and pathological processes. Active MMPs are a family of single-chain enzymes (23 family members in the human genome), most of which constituted by a catalytic domain and by a hemopexin-like domain connected by a linker. The X-ray structures of MMP-1 and MMP-2 suggest a conserved and well-defined spatial relationship between the two domains. Here we present structural data for MMP-12, suitably stabilized against self-hydrolysis, both in solution (NMR and SAXS) and in the solid state (X-ray), showing that the hemopexin-like and the catalytic domains experience conformational freedom with respect to each other on a time scale shorter than 10(-8) s. Hints on the probable conformations are also obtained. This experimental finding opens new perspectives for the often hypothesized active role of the hemopexin-like domain in the enzymatic activity of MMPs.

Molecular recognition and substrate mimicry drive the electron-transfer process between MIA40 and ALR

Oxidative protein folding in the mitochondrial intermembrane space requires the transfer of a disulfide bond from MIA40 to the substrate. During this process MIA40 is reduced and regenerated to a functional state through the interaction with the flavin-dependent sulfhydryl oxidase ALR. Here we present the mechanistic basis of ALR–MIA40 interaction at atomic resolution by biochemical and structural analyses of the mitochondrial ALR isoform and its covalent mixed disulfide intermediate with MIA40. This ALR isoform contains a folded FAD-binding domain at the C-terminus and an unstructured, flexible N-terminal domain, weakly and transiently interacting one with the other. A specific region of the N-terminal domain guides the interaction with the MIA40 substrate binding cleft (mimicking the interaction of the substrate itself), without being involved in the import of ALR. The hydrophobicity-driven binding of this region ensures precise protein–protein recognition needed for an efficient electron transfer process.

Copper(I)-mediated protein-protein interactions result from suboptimal interaction surfaces.

The homoeostasis of metal ions in cells is the result of the contribution of several cellular pathways that involve transient, often weak, protein-protein interactions. Metal transfer typically implies the formation of adducts where the metal itself acts as a bridge between proteins, by co-ordinating residues of both interacting partners. In the present study we address the interaction between the human copper(I)-chaperone HAH1 (human ATX1 homologue) and a metal-binding domain in one of its partners, namely the P-type copper-transporting ATPase, ATP7A (ATPase, Cu+ transporting, alpha polypeptide). The adduct was structurally characterized in solution, in the presence of copper(I), and through X-ray crystallography, upon replacing copper(I) with cadmium(II). Further insight was obtained through molecular modelling techniques and site-directed mutagenesis. It was found that the interaction involves a relatively small interface (less than 1000 A(2), 1 A=0.1 nm) with a low fraction of non-polar atoms. These observations provide a possible explanation for the low affinity of the two apoproteins. It appears that electrostatics is important in selecting which domain of the ATPase is able to form detectable amounts of the metal-mediated adduct with HAH1.

Structural basis of serine/threonine phosphatase inhibition by the archetypal small molecules cantharidin and norcantharidin

The inhibition of a subgroup of human serine/threonine protein phosphatases is responsible for the cytotoxicity of cantharidin and norcantharidin against tumor cells. It is shown that the anhydride rings of cantharidin and norcantharidin are hydrolyzed when bound to the catalytic domain of the human serine/threonine protein phosphatases 5 (PP5c), and the high-resolution crystal structures of PP5c complexed with the corresponding dicarboxylic acid derivatives of the two molecules are reported. Norcantharidin shows a unique binding conformation with the catalytically active Mn2PP5c, while cantharidin is characterized by a double conformation in its binding mode to the protein. Different binding modes of norcantharidin are observed depending of whether the starting ligand is in the anhydride or in the dicarboxylic acid form. All these structures will provide the basis for the rational design of new cantharidin-based drugs.

Entropic contribution to the linking coefficient in fragment based drug design: a case study.

For several drug leads obtained by tethering weak binding ligands, the dissociation constant is smaller than the product of those of the individual fragments by a factor named the linking coefficient, E. This favorable contribution is attributed to the entropic gain that is realized when two weak binding ligands are tethered. Here we show a case study where the linking coefficient is strikingly small (E = 2.1 x 10(-3) M(-1)) and its totally entropic nature is demonstrated.

Interaction of cisplatin with human superoxide dismutase.

cis-Diamminedichloroplatinum(II) (cisplatin) is able to interact with human superoxide dismutase (hSOD1) in the disulfide oxidized apo form with a dissociation constant of 37 ± 3 μM through binding cysteine 111 (Cys111) located at the edge of the subunit interface. It also binds to Cu(2)-Zn(2) and Zn(2)-Zn(2) forms of hSOD1. Cisplatin inhibits aggregation of demetalated oxidized hSOD1, and it is further able to dissolve and monomerize oxidized hSOD1 oligomers in vitro and in cell, thus indicating its potential as a leading compound for amyotrophic lateral sclerosis.

High-Resolution Crystal Structure of the Subclass B3 Metallo-β-Lactamase BJP-1: Rational Basis for Substrate Specificity and Interaction with Sulfonamides

ABSTRACT Metallo-β-lactamases (MBLs) are important enzymatic factors in resistance to β-lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes β-lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for β-lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.

Molecular view of an electron transfer process essential for iron–sulfur protein biogenesis

Biogenesis of iron–sulfur cluster proteins is a highly regulated process that requires complex protein machineries. In the cytosolic iron–sulfur protein assembly machinery, two human key proteins—NADPH-dependent diflavin oxidoreductase 1 (Ndor1) and anamorsin—form a stable complex in vivo that was proposed to provide electrons for assembling cytosolic iron–sulfur cluster proteins. The Ndor1–anamorsin interaction was also suggested to be implicated in the regulation of cell survival/death mechanisms. In the present work we unravel the molecular basis of recognition between Ndor1 and anamorsin and of the electron transfer process. This is based on the structural characterization of the two partner proteins, the investigation of the electron transfer process, and the identification of those protein regions involved in complex formation and those involved in electron transfer. We found that an unstructured region of anamorsin is essential for the formation of a specific and stable protein complex with Ndor1, whereas the C-terminal region of anamorsin, containing the [2Fe-2S] redox center, transiently interacts through complementary charged residues with the FMN-binding site region of Ndor1 to perform electron transfer. Our results propose a molecular model of the electron transfer process that is crucial for understanding the functional role of this interaction in human cells.