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Understanding the fine structural details of inhibitor binding at the active site of metalloenzymes can have a profound impact on the rational drug design targeted to this broad class of biomolecules. Structural techniques such as NMR, cryo-EM, and X-ray crystallography can provide bond lengths and angles, but the uncertainties in these measurements can be as large as the range of values that have been observed for these quantities in all the published structures. This uncertainty is far too large to allow for reliable calculations at the quantum chemical (QC) levels for developing precise structure–activity relationships or for improving the energetic considerations in protein-inhibitor studies. Therefore, the need arises to rely upon computational methods to refine the active site structures well beyond the resolution obtained with routine application of structural methods. In a recent paper, we have shown that it is possible to refine the active site of cobalt(II)-substituted MMP12, a metalloprotein that is a relevant drug target, by matching to the experimental pseudocontact shifts (PCS) those calculated using multireference ab initio QC methods. The computational cost of this methodology becomes a significant bottleneck when the starting structure is not sufficiently close to the final one, which is often the case with biomolecular structures. To tackle this problem, we have developed an approach based on a neural network (NN) and a support vector regression (SVR) and applied it to the refinement of the active site structure of oxalate-inhibited human carbonic anhydrase 2 (hCAII), another prototypical metalloprotein target. The refined structure gives a remarkably good agreement between the QC-calculated and the experimental PCS. This study not only contributes to the knowledge of CAII but also demonstrates the utility of combining machine learning (ML) algorithms with QC calculations, offering a promising avenue for investigating other drug targets and complex biological systems in general.

Oxaliplatin (OXA) is a valuable and largely used cancer drug which induces a serious and intractable neuropathy. The lipoyl-homotaurine derivative (ADM_12) reverts in vivo OXA-induced neuropathy, and it is an effective antagonist of the nociceptive sensor channel TRPA1. Unprecedentedly, this safe analgesic showed a synergy with OXA in vitro and proved to inhibit CA IX, a relevant therapeutic target, clearly interfering with pancreatic cancer cells’ aggressiveness.

The maturation of mitochondrial iron-sulfur proteins requires a complex protein machinery. Human IBA57 protein was proposed to act in a late phase of this machinery, along with GLRX5, ISCA1, and ISCA2. However, a molecular picture on how these proteins cooperate is not defined yet. We show here that IBA57 forms a heterodimeric complex with ISCA2 by bridging a [2Fe-2S] cluster, that [2Fe-2S] cluster binding is absolutely required to promote the complex formation, and that the cysteine of the conserved motif characterizing IBA57 protein family and the three conserved cysteines of the ISCA protein family act as cluster ligands. The [2Fe-2S] heterodimeric complex is the final product when IBA57 is either exposed to [2Fe-2S] ISCA2 or in the presence of [2Fe-2S] GLRX5 and apo ISCA2. We also find that the [2Fe-2S] ISCA2-IBA57 complex is resistant to highly oxidative environments and is capable of reactivating apo aconitase in vitro. Collectively, our data delinate a [2Fe-2S] cluster transfer pathway involving three partner proteins of the mitochondrial ISC machinery, that is, GLRX5, ISCA2 and IBA57, which leads to the formation of a [2Fe-2S] ISCA2-IBA57 complex.

In-cell NMR spectroscopy is a powerful approach to study protein structure and function in the native cellular environment. It provides precious insights into the folding, maturation, interactions, and ligand binding of important pharmacological targets directly in human cells. However, its widespread application is hampered by the fact that soluble globular proteins often interact with large cellular components, causing severe line broadening in conventional heteronuclear NMR experiments. 19F NMR can overcome this issue, as fluorine atoms incorporated in proteins can be detected by simple background-free 1D NMR spectra. Here, we show that fluorinated amino acids can be easily incorporated in proteins expressed in human cells by employing a medium switch strategy. This straightforward approach allows the incorporation of different fluorinated amino acids in the protein of interest, reaching fluorination efficiencies up to 60%, as confirmed by mass spectrometry and X-ray crystallography. The versatility of the approach is shown by performing 19F in-cell NMR on several proteins, including those that would otherwise be invisible by 1H-15N in-cell NMR. We apply the approach to observe the interaction between an intracellular target, carbonic anhydrase 2, and its inhibitors, and to investigate how the formation of a complex between superoxide dismutase 1 and its chaperone CCS modulates the interaction of the chaperone subunit with the cellular environment.

ABSTRACTMetallo-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 japonicumthat 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.

ABSTRACTClass 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 aeruginosaclinical 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.

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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, 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.

Human α-lactalbumin has not been described as a glycoprotein, despite the fact that several α-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 α-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 α-lactalbumins except red-necked wallaby. That a relatively small proportion of the protien is glycosylated (about 1%) may reflect the importance of this region of the protein sequence to the molten globule state of α-lactalbumin.

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