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Malate dehydrogenases (MDHs) sustain tumor growth and carbon metabolism by pathogens including Plasmodium falciparum. However, clinical success of MDH inhibitors is absent, as current small molecule approaches targeting the active site are unselective. The presence of an allosteric binding site at oligomeric interface allows the development of more specific inhibitors. To this end we performed a differential NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface. Subsequent biophysical and biochemical experiments of an identified fragment indicate an allosteric mechanism of 4-(3,4-difluorophenyl) thiazol-2-amine (4DT) inhibition by impacting the formation of the active site loop, located >30 Å from the 4DT binding site. Further characterization of the more tractable homolog 4-phenylthiazol-2-amine (4PA) and 16 other derivatives are also reported. These data pave the way for downstream development of more selective molecules by utilizing the oligomeric interfaces showing higher species sequence divergence than the MDH active site., Romero et al. perform NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface of malate dehydrogenase (MDH). Their study indicates an allosteric mechanism impacting enzymatic activity, paving the way for development of more selective molecules and a starting point for the future development of specific MDH inhibitors.

Partial symmetry, i.e., the presence of more than one molecule in the asymmetric unit of a crystal, is a relatively rare phenomenon in small-molecule crystallography, but is quite common in protein crystallography, where it is typically known as non-crystallographic symmetry (NCS). Several papers in literature propose molecular determinants such as crystal contacts, thermal factors, or TLS parameters as an explanation for the phenomenon of intrinsic asymmetry among molecules that are in principle equivalent. Nevertheless, are all of the above determinants the cause or are they rather the effect? In the general frame of the NCS often observed in crystals of biomolecules, this paper deals with nickel(II)-substituted human carbonic anhydrase(II) (hCAII) and its SAD structure determination at the nickel edge. The structure revealed two non-equivalent molecules in the asymmetric unit, the presence of a secondary nickel-binding site at the N-terminus of both molecules (which had never been found before in the nickel-substituted enzyme) and two different coordination geometries of the active site nickel (hexa-coordinated in one molecule and mainly penta-coordinated in the other). The above-mentioned standard molecular crystallographic determinants of this asymmetry are analyzed and presented in detail for this particular case. From these considerations, we speculate on the existence of a fundamental, although yet unknown, common cause for the partial symmetry that is so often encountered in X-ray structures of biomolecules.

The oxytocinase subfamily of M1 aminopeptidases consists of three members, ERAP1, ERAP2 and IRAP that play several important biological roles, including key functions in the generation of antigenic peptides that drive human immune responses. They represent emerging targets for pharmacological manipulation of the immune system, albeit lack of selective inhibitors is hampering these efforts. Most of the previously explored small-molecule binders target the active site of the enzymes via strong interactions with the catalytic zinc(II) atom and, while achieving increased potency, they suffer in selectivity. Continuing our earlier efforts on weaker zinc(II) binding groups (ZBG), like the 3,4-diaminobenzoic acid derivatives (DABA), we herein synthesized and biochemically evaluated analogues of nine potentially weak ZBGs, based on differential substitutions of functionalized pyridinone- and pyridinethione-scaffolds, nicotinic-, isonicotinic-, aminobenzoic- and hydrazinobenzoic-acids. Crystallographic analysis of two analogues in complex with a metalloprotease (MMP-12) revealed unexpected binding topologies, consistent with the observed affinities. Our results suggest that the potency of the compounds as inhibitors of ERAP1, ERAP2 and IRAP is primarily driven by the occupation of active-site specificity pockets and their proper orientation within the enzymes.

[Image: see text] The formation of amorphous protein aggregates containing human superoxide dismutase (hSOD1) is thought to be involved in amyotrophic lateral sclerosis onset. cis-Platin inhibits the oligomerization of apo hSOD1, but its toxicity precludes any possible use in therapy. Herein, we propose a less toxic platinum complex, namely oxa/cis-platin, as hSOD1 antiaggregation lead compound. Oxa/cis-platin is able to interact with hSOD1 in the disulfide oxidized apo form by binding cysteine 111 (Cys111). The mild neurotoxic phenomena induced in vitro and in vivo by oxa/cis-platin can be successfully reverted by using lypoyl derivatives, which do not interfere with the antiaggregation properties of the platin derivative.

Ind1, a mitochondrial P-loop NTPase is essential for assembly of respiratory complex-I. Respiratory complex-I (NADH: ubiquinone oxidoreductase), a large (mitochondrial inner membrane) enzyme, is made of 45 subunits and 8 iron-sulfur clusters. Ind1, an iron-sulfur cluster protein involved in the maturation of respiratory complex and binds an Fe/S cluster via a conserved CXXC motif in a labile way. Ind1 has been proposed as a specialized biogenesis factor involved in delivering the Fe/S clusters to the apo complex-I subunits. The IND1 gene is conserved in eukaryotes and is present in genomes of the species that retain functional respiratory complex-I. Depletion of human Ind1 causes ultra-structural changes in depleted mitochondria, including the loss of cristae membranes, massive remodeling of respiratory super complexes, and increased lactate production. Ind1 sequence bears known nucleotide binding domain motifs and was first classified as Nucleotide Binding Protein-Like (NUBPL). Despite the obvious importance of Ind1, very little is known about this protein; in particular its structure as well as its Fe/S cluster binding properties. In the present work we show that the expression of native huInd1 in Escherichia coli stimulates over-expression of the beta-lactamase TEM-1 from E. coli. The homology modeling of huInd1 shows hallmark of Rossmann fold, where a central beta sheet is covered by helices on either side. In the light of the modeled structure of huInd1, we hypothesize that huInd1 binds to the untranslated region (UTR) of the TEM-1 mRNA at 3' site and thereby reducing the possibility of its endonucleolytic cleavage, resulting in over-expression of TEM-1.

Paramagnetic NMR data (pseudocontact shifts and self-orientation residual dipolar couplings) and diamagnetic residual dipolar couplings can now be used in the program REFMAC5 from CCP4 as structural restraints together with X-ray crystallographic data. These NMR restraints can reveal differences between solid state and solution conformations of molecules or, in their absence, can be used together with X-ray crystallographic data for structural refinement., The program REFMAC5 from CCP4 was modified to allow the simultaneous use of X-ray crystallographic data and paramagnetic NMR data (pseudocontact shifts and self-orientation residual dipolar couplings) and/or diamagnetic residual dipolar couplings. Incorporation of these long-range NMR restraints in REFMAC5 can reveal differences between solid-state and solution conformations of molecules or, in their absence, can be used together with X-ray crystallographic data for structural refinement. Since NMR and X-ray data are complementary, when a single structure is consistent with both sets of data and still maintains reasonably ‘ideal’ geometries, the reliability of the derived atomic model is expected to increase. The program was tested on five different proteins: the catalytic domain of matrix metalloproteinase 1, GB3, ubiquitin, free calmodulin and calmodulin complexed with a peptide. In some cases the joint refinement produced a single model consistent with both sets of observations, while in other cases it indicated, outside the experimental uncertainty, the presence of different protein conformations in solution and in the solid state.

The molecular determinants responsible for the potency of the RXP470.1 phosphinic 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/inhibitor complex (1.15 Å) reveals that the inhibitor establishes multiple interactions with the MMP-12 active site, with its long P(1)' side chain filling most of the S(1)' deep cavity. ITC experiments indicate that the binding of this inhibitor to MMP-12 is mostly entropy driven (ΔG° = -13.1 kcal/mol, ΔH° = -2.53 kcal/mol, and -TΔS° = -10.60 kcal/mol) and involves a proton uptake from the buffer. Comparing phosphinic versus hydroxamate inhibitors reveals that the chelation of the zinc ion is slightly different, leading the inhibitor backbone to adopt a position in which the hydrogen bonding with the MMP-12 active site is less favorable in phosphinic inhibitor while maintaining high affinity.

TEM-72, a class A β-lactamase identified in isolates of Enterobacteriaceae, is a quadruple mutant of TEM-1 (Q39K, M182T, G238S and E240K) and shows extended-spectrum β-lactamase (ESBL) properties arising from the G238S and E240K substitutions. Although many structures of TEM variants have been published, they do not include an enzyme with the simultaneous presence of both of the ESBL-conferring G238S and E240K substitutions. Furthermore, the structure shows the presence of a citrate anion bound to the TEM-72 active site, where it interacts with all of the conserved residues of class A β-lactamases. The present structure supports the use of polycarboxylates as a scaffold for the design of broad-spectrum inhibitors of serine β-lactamases.

N-arylsulfonyl-based MMPs inhibitors (MMPIs) are among the most prominent inhibitors possessing nanomolar affinity. However, their poor bioavailability remains critical for the drug development of this family of molecules. The structural analysis of the complex of NNGH (the most representative member of the family) with MMP-12 provided us with the basis to effectively design simple NNGH analogues with enhanced solubility in water. Following this approach, the sec-butyl residue, not directly involved in the binding with MMP, has been replaced with hydrophilic residues thus yielding new potent inhibitors soluble in water.

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, α 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 Å2, 1 Å=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.

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.

The solution structures of apo, Cu(I), and Ni(II) human Sco1 have been determined. The protein passes from an open and conformationally mobile state to a closed and rigid conformation upon metal binding as shown by electrospray ionization MS and NMR data. The metal ligands of Cu(I) are two Cys residues of the CPXXCP motif and a His residue. The latter is suitably located to coordinate the metal anchored by the two Cys residues. The coordination sphere of Ni(II) in solution is completed by another ligand, possibly Asp. Crystals of the Ni(II) derivative were also obtained with the Ni(II) ion bound to the same His residue and to the two oxidized Cys residues of the CPXXCP motif. We propose that the various structures solved here represent the various states of the protein in its functional cycle and that the metal can be bound to the oxidized protein at a certain stage. Although it now seems reasonable that Sco1, which is characterized by a thioredoxin fold, has evolved to bind a metal atom via the di-Cys motif to act as a copper chaperone, the oxidized form of the nickel-bound protein suggests that it may also maintain the thioredoxin function.

The smallest extrinsic polypeptide of the water-oxidizing complex (PsbQ) was extracted and purified from spinach (Spinacia oleracea) photosystem II (PSII) membranes. It was then crystallized in the presence of Zn2+ and its structure was determined by X-ray diffraction at 1.95-Å resolution using the multi-wavelength anomalous diffraction method, with the zinc as the anomalous scatterer. The crystal structure shows that the core of the protein is a four-helix bundle, whereas the amino-terminal portion, which possibly interacts with the photosystem core, is not visible in the crystal. The distribution of positive and negative charges on the protein surface might explain the ability of PsbQ to increase the binding of Cl− and Ca2+ and make them available to PSII.

Cellular retinol-binding proteins (CRBPs) are cytoplasmic retinol-specific binding proteins. Mammalian CRBPs have been thoroughly characterised previously. Here we report on the identification and X-ray structural analysis of the apo (1.7A resolution) and holo (1.4A resolution) forms of a zebrafish CRBP. According to amino acid sequence and structure analyses, the zebrafish CRBP that we have identified resembles closely mammalian CRBP II, suggesting that it is the zebrafish orthologue of this mammalian CRBP type. Zebrafish CRBP forms a tight complex with all-trans retinol, producing an absorption spectrum similar to those of mammalian holo-CRBPs, albeit slightly blue-shifted. The superposition of the alpha-carbon atoms of the liganded (complexed with retinol) and unliganded forms of zebrafish CRBP shows significant differences in correspondence of the betaC-betaD (residues 55-58) and betaE-betaF (residues 74-77) turns, providing evidence for the occurrence of conformational changes accompanying retinol binding/release. Remarkable and well-defined ligand-dependent conformational changes in the protein region comprising the two beta-turns affect both the main chain and the side-chains of several residues. The two beta-turns project towards the interior of the cavity devoid of ligand of the apoprotein. The side-chains of F57, Y60 and L77 change substantially their orientation and position in the apoprotein relative to the holoprotein. In the beta-barrel internal cavity of apo-CRBP they occupy some of the space that is otherwise occupied by bound retinol in holo-CRBP, and are displaced from these positions on ligand binding. These results indicate that a flexible area encompassing the betaC-betaD and betaE-betaF turns may serve as the ligand portal and that these turns undergo conformational changes associated with the not yet clarified mechanism of retinol binding and release in CRBPs.

Human alpha-lactalbumin has not been described as a glycoprotein, despite the fact that several alpha-lactalbumins of both ruminant and nonruminant species are known to be glycosylated. In all these species the glycosylation site is the 45Asn in the usual triplet 45Asn-Gly/Gln-47Ser. We have found that human alpha-lactalbumin is glycosylated and the glycosylation site has been determined by protein sequencing and mass spectrometry. We report an unusual glycosylation site at 71Asn in the triplet 71Asn-Ile-73Cys, which is conserved in all known alpha-lactalbumins except red-necked wallaby. That a relatively small proportion of the protein is glycosylated (about 1%) may reflect the importance of this region of the protein sequence to the molten globule state of alpha-lactalbumin.

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.

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.

The oxidative folding mechanism in the intermembrane space of human mitochondria underpins a disulfide relay system consisting of the import receptor Mia40 and the homodimeric FAD-dependent thiol oxidase ALR. The flavoprotein ALR receives two electrons per subunit from Mia40, which are then donated through one-electron reactions to two cytochrome c molecules, thus mediating a switch from two-electron to one-electron transfer. We dissect here the mechanism of the electron flux within ALR, characterizing at the atomic level the ALR intermediates that allow electrons to rapidly flow to cytochrome c. The intermediate critical for the electron-transfer process implies the formation of a specific inter-subunit disulfide which exclusively allows electron flow from Mia40 to FAD. This finding allows us to present a complete model for the electron-transfer pathway in 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.

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.

Class D β-lactamases represent a heterogeneous group of active-site serine β-lactamases that show an extraordinary panel of functional features and substrate profiles, thus representing relevant models for biochemical and structural studies. OXA-46 is a narrow-spectrum enzyme belonging to the OXA-2 subgroup which was found in a Pseudomonas aeruginosa clinical isolate from northern Italy. In this work, we obtained the three-dimensional structure of OXA-46, which shows the overall fold of active serine β-lactamases and a dimeric quaternary structure. Significant differences with currently available structures of class D β-lactamases were found in the loops located close to the active site, which differ in length and conformation. Interestingly, the three subunits present in the asymmetric unit showed some structural heterogeneity, only one of which presented a carbamylated lysine recognized as an important functional feature of class D enzymes. The carbamylation state of residue Lys75 appeared to be associated with different shapes and dimensions of the active site. Moreover, a tartrate molecule from the crystallization buffer was found in the active site of the noncarbamylated subunits, which interacts with catalytically relevant residues. The OXA-46 crystal asymmetric units thus interestingly present the structures of the free carbamylated active site and of the ligand-bound uncarbamylated active site, offering the structural basis for investigating the potential of new scaffolds of β-lactamase inhibitors.

The design and synthesis of biotin chain-terminated inhibitors (BTI) showing high affinity for matrix metalloproteinases (MMPs) on one side and high affinity for avidin through the biotinylated tag on the other are reported. The affinity of the designed BTI toward five different MMPs has been evaluated and the simultaneous formation of a highly stable ternary system Avidin-BTI-MMP clearly assessed. This system will permit the development of new approaches to detect, quantify, or collect MMPs in biological samples, with potential applications in vivo.

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.

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.

Little is known about prokaryotic homologs of Cu,Zn superoxide dismutase (SOD), an enzyme highly conserved among eukaryotic species. In 138 Archaea and Bacteria genomes, 57 of these putative homologs were found, 11 of which lack at least one of the metal ligands. Both the solution and the crystal structures of the SOD-like protein from Bacillus subtilis , lacking two Cu ligands and found to be enzymatically inactive, were determined. In solution, the protein is monomeric. The available nuclear Overhauser effects, together with chemical-shift index values, allowed us to define and to recognize the typical Cu,Zn SOD Greek β-barrel but with largely unstructured loops (which, therefore, sample a wide range of conformations). On the contrary, in the crystal structure (obtained in the presence of slight excess of Zn), the protein is well structured and organized in covalent dimers held by a symmetric bridge consisting of a Zn ion bound to an Asp–His dyad in a tetrahedral geometry. Couples of dimers held by hydrophobic interactions and H bonds are further organized in long chains. The order/disorder transition is discussed in terms of metal binding and physical state.

It is known that several prokaryotic protein sequences, characterized by high homology with the eukaryotic Cu,ZnSODs, lack some of the metal ligands. In the present work, we have stepwise reintroduced the two missing copper ligands in the SOD-like protein of Bacillus subtilis, through site-directed mutagenesis. The mutant with three out of the four His that bind copper is not active, whereas the fully reconstituted mutant displays an activity of about 10% that of human Cu,ZnSOD. The mutated proteins have been characterized in solution and in the solid state. In solution, the proteins experience conformational disorder, which is believed to be partly responsible for the decreased enzymatic activity and sheds light on the tendency of several human SOD mutants to introduce mobility in the protein frame. In the crystal, on the contrary, the protein has a well-defined conformation, giving rise to dimers through the coordination of an exogenous zinc ion. The catalytic properties of the double mutant, which might be regarded as a step in an artificial evolution from a nonactive SOD to a fully functioning enzyme, are discussed on the basis of the structural and dynamical properties.

Copper-containing amine oxidase extracted from bovine serum (BSAO) was crystallized and its three-dimensional structure at 2.37 A 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.

AphA is a periplasmic acid phosphatase of Escherichia coli belonging to class B bacterial phosphatases, which is part of the DDDD superfamily of phosphohydrolases. The crystal structure of AphA has been determined at 2.2 A and its resolution extended to 1.7 A on an AuCl 3 derivative. This represents the first crystal structure of a class B bacterial phosphatase. Despite the lack of sequence homology, the AphA structure reveals a haloacid dehalogenase-like fold. This finding suggests that this fold could be conserved among members of the DDDD superfamily of phosphohydrolases. The active enzyme is a homotetramer built by using an extended N-terminal arm intertwining the four monomers. The active site of the native enzyme, as prepared, hosts a magnesium ion, which can be replaced by other metal ions. The structure explains the non-specific behaviour of AphA towards substrates, while a structure-based alignment with other phosphatases provides clues about the catalytic mechanism.

CutA1 are a protein family present in bacteria, plants, and animals, including humans. Escherichia coli CutA1 is involved in copper tolerance, whereas mammalian proteins are implicated in the anchoring of acetylcholinesterase in neuronal cell membranes. The x-ray structures of CutA1 from E. coli and rat were determined. Both proteins are trimeric in the crystals and in solution through an inter-subunit beta-sheet formation. Each subunit consists of a ferredoxin-like (beta1alpha1beta2beta3alpha2beta4) fold with an additional strand (beta5), a C-terminal helix (alpha3), and an unusual extended beta-hairpin involving strands beta2 and beta3. The bacterial CutA1 is able to bind copper(II) in vitro through His2Cys coordination in a type II water-accessible site, whereas the rat protein precipitates in the presence of copper(II). The evolutionarily conserved trimeric assembly of CutA1 is reminiscent of the architecture of PII signal transduction proteins. This similarity suggests an intriguing role of CutA1 proteins in signal transduction through allosteric communications between subunits.

The class B non-specific acid phosphatase AphA from Escherichia coli has been expressed in E. coli and purified following a new protocol. ESI mass spectroscopy shows that the purified enzyme solution contains two polypeptides with molecular weights differing by 185 Da corresponding to two different cleavage sites of the signal peptide from the AphA E. coli precursor. Despite the solution heterogeneity, X-ray quality crystals have been obtained. However, the crystals have a tendency to give polymorphs and to lose long-range order with time while maintaining an intact crystal habit. Crystals have been grown in space groups I222 and C2 with three different unit cells and different asymmetric unit contents. Diffraction data to 1.6 A resolution have been collected with synchrotron radiation at ESRF and DESY.

A copper-containing amine oxidase extracted from bovine serum (BSAO) and purified to homogeneity has been deglycosylated and crystallized. The crystals obtained belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 77.68, b = 131.19, c = 134.00 A, and diffract to at least 2.4 A resolution. BSAO is the first mammalian amine oxidase to be crystallized.

Three cellular retinol-binding protein (CRBP) types (CRBP I, II, and III) with distinct tissue distributions and retinoid binding properties have been structurally characterized thus far. A human binding protein, whose mRNA is expressed primarily in kidney, heart, and transverse colon, is shown here to be a CRBP family member (human CRBP IV), according to amino acid sequence, phylogenetic analysis, gene structure organization, and x-ray structural analysis. Retinol binding to CRBP IV leads to an absorption spectrum distinct from a typical holo-CRBP spectrum and is characterized by an affinity (K(d) = approximately 200 nm) lower than those for CRBP I, II, and III, as established in direct and competitive binding assays. As revealed by mutagenic analysis, the presence in CRBP IV of His(108) in place of Gln(108) is not responsible for the unusual holo-CRBP IV spectrum. The 2-A resolution crystal structure of human apo-CRBP IV is very similar to those of other structurally characterized CRBPs. The side chain of Tyr(60) is present within the binding cavity of the apoprotein and might affect the interaction with the retinol molecule. These results indicate that human CRBP IV belongs to a clearly distinct CRBP subfamily and suggest a relatively different mode of retinol binding for this binding protein.

3-Hydroxyanthranilic acid oxygenase (3-HAO) catalyses the conversion of 3-hydroxyanthranilic acid to quinolinic acid. Because of the involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we have cloned human 3-HAO in Escherichia coli , overexpressed and purified it with the aim of studying its enzymatic activity and for future structural studies. The recombinant human protein, obtained in E. coli , retains its enzymatic activity which can occur only in the presence of Fe(II); several other metals have been tested but in no case the formation of the product has been observed. On the contrary, two of the ions tested inhibit the catalytic reaction and one of them, Zn 2+ , could be of physiological relevance. A circular dichroism analysis has also been performed, showing that the secondary structure is mainly of the β type, with a minority of α.

The crystal structure of the specific carrier of retinol (retinol-binding protein, RBP) purified from chicken plasma has been determined (space group P2(1)2(1)2(1), with a=46.06(5) A, b=53.56(6) A, c=73.41(8) A, and one protein molecule in the asymmetric unit). Despite being obtained from a species phylogenetically distant from mammals, chicken holoRBP has an overall structure that closely resembles the previously determined structures of mammalian holoRBPs. The lack in chicken RBP of eight carboxy-terminal amino acid residues characteristic of mammalian RBPs does not significantly affect the protein structure. A distinctive feature of the avian protein is a better definition of the loop 63-67, close to the opening of the beta-barrel cavity accommodating the retinol molecule, which is rather disordered in the structures of mammalian RBPs.

Two cellular retinol-binding proteins (CRBP I and II) with distinct tissue distributions and retinoid-binding properties have been recognized thus far in mammals. Here, we report the identification of a human retinol-binding protein resembling type I (55.6% identity) and type II (49.6% identity) CRBPs, but with a unique H residue in the retinoid-binding site and a distinctively different tissue distribution. Additionally, this binding protein (CRBP III) exhibits a remarkable sequence identity (62.2%) with the recently identified ι-crystallin/CRBP of the diurnal gecko Lygodactylus picturatus [Werten, P. J. L., Röll, B., van Alten, D. M. F. & de Jong, W. W. (2000) Proc. Natl. Acad. Sci. USA 97, 3282–3287 (First Published March 21, 2000; 10.1073/pnas.050500597)]. CRBP III and all-trans -retinol form a complex ( K d ≈ 60 nM), the absorption spectrum of which is characterized by the peculiar fine structure typical of the spectra of holo-CRBP I and II. As revealed by a 2.3-Å x-ray molecular model of apo-CRBP III, the amino acid residues that line the retinol-binding site in CRBP I and II are positioned nearly identically in the structure of CRBP III. At variance with the human CRBP I and II mRNAs, which are most abundant in ovary and intestine, respectively, the CRBP III mRNA is expressed at the highest levels in kidney and liver thus suggesting a prominent role for human CRBP III as an intracellular mediator of retinol metabolism in these tissues.

Isolation, purification, amino acid sequence determination and X-ray crystal structure of buffalo alpha-lactalbumin were performed in order to gain further knowledge of the molecular basis of alpha-lactalbumin in the lactose synthase complex. The deduced amino acid sequence differs at one position from the bovine alpha-lactalbumin sequence (at position 17). The refined crystal structure at 2.3 A is very similar to those previously reported for human and baboon alpha-lactalbumins. However, a portion of the molecule (residues 105-109) exhibits different conformation. It forms a 'flexible loop', and appears to be a functionally important region in forming the lactose synthase complex.

It is widely believed that cow's milk proteins ingested by the mother, in particular beta-lactoglobulin (beta-LG), can pass into breast milk and thus sensitize predisposed infants. However, studies to evaluate bovine beta-LG in human milk have given conflicting results. The aim of this study was to analyse the correlation between the amount of cow's milk in the mother's diet and the presence of bovine beta-LG in breast milk. Human milk samples from 14 healthy non-atopic women on diets with different cow's milk contents were examined. The total concentration of bovine beta-LG or beta-LG immuno-like proteins (beta-LGIP) was determined by enzyme-linked immunosorbent assay (ELISA). Two separation procedures utilizing ELISA plates and an affinity chromatography column were set up to identify the human whey components recognized by the anti-beta-LG antibodies. beta-LGIP reactivities of milk from three groups on different diets were not significantly different. After splitting the antigen-antibody complexes, three main protein components, human lactoferrin, human beta-casein and human alpha-lactalbumin, were identified. This study would suggest that, at least in healthy subjects, false-positive results in ELISA determinations of bovine beta-LG in human milk might be due to cross-reactions between polyclonal antibodies and different protein antigens.