Daniele de Sanctis

ID29: a high-intensity highly automated ESRF beamline for macromolecular crystallography experiments exploiting anomalous scattering.

ID29 is an ESRF undulator beamline with a routinely accessible energy range of between 20.0 keV and 6.0 keV (λ = 0.62 Å to 2.07 Å) dedicated to the use of anomalous dispersion techniques in macromolecular crystallography. Since the beamline was first commissioned in 2001, ID29 has, in order to provide an improved service to both its academic and proprietary users, been the subject of almost continuous upgrade and refurbishment. It is now also the home to the ESRF Cryobench facility, ID29S. Here, the current status of the beamline is described and plans for its future are briefly outlined.

Reversible hexa- to penta-coordination of the heme Fe atom modulates ligand binding properties of neuroglobin and cytoglobin.

Neuroglobin (Ngb) and cytoglobin (Cygb) are two recently discovered intracellular members of the vertebrate hemoglobin (Hb) family. Ngb, predominantly expressed in nerve cells, is of ancient evolutionary origin and is homologous to nerve‐globins of invertebrates. Cygb, present in many different tissues, shares common ancestry with myoglobin (Mb) and can be traced to early vertebrate evolution. Ngb is held to facilitate O2 diffusion to the mitochondria and to protect neuronal cells from hypoxic‐ischemic insults, may be an oxidative stress‐responsive sensor protein for signal transduction, and may carry out enzymatic activities, such as NO/O2 scavenging. Cygb is linked to collagen synthesis, may provide O2 for enzymatic reactions, and may be involved in a ROS (NO)‐signaling pathway(s). Ngb and Cgb display the classical three‐over‐three α‐helical fold of Hb and Mb, and are endowed with a hexa‐coordinate heme‐Fe atom, in their ferrous and ferric forms, having the heme distal HisE7 residue as the endogenous ligand. Reversible hexa‐ to penta‐coordination of the heme Fe atom modulates ligand binding properties of Ngb and Cygb. Moreover, Ngb and Cygb display a tunnel/cavity system within the protein matrix held to facilitate ligand channeling to/from the heme, multiple ligand copies storage, multi‐ligand reactions, and conformational transitions supporting ligand binding. IUBMB Life, 56: 657‐664, 2004

New Evidence for the Role of Calcium in the Glycosidase Reaction of Gh43 Arabinanases.

Endo‐1,5‐α‐l‐arabinanases are glycosyl hydrolases that are able to cleave the glycosidic bonds of α‐1,5‐l‐arabinan, releasing arabino‐oligosaccharides and l‐arabinose. Two extracellular endo‐1,5‐α‐l‐arabinanases have been isolated from Bacillus subtilis, BsArb43A and BsArb43B (formally named AbnA and Abn2, respectively). BsArb43B shows low sequence identity with previously characterized 1,5‐α‐l‐arabinanases and is a much larger enzyme. Here we describe the 3D structure of native BsArb43B, biochemical and structure characterization of two BsArb43B mutant proteins (H318A and D171A), and the 3D structure of the BsArb43B D171A mutant enzyme in complex with arabinohexose. The 3D structure of BsArb43B is different from that of other structurally characterized endo‐1,5‐α‐l‐arabinanases, as it comprises two domains, an N‐terminal catalytic domain, with a 3D fold similar to that observed for other endo‐1,5‐α‐l‐arabinanases, and an additional C‐terminal domain. Moreover, this work also provides experimental evidence for the presence of a cluster containing a calcium ion in the catalytic domain, and the importance of this calcium ion in the enzymatic mechanism of BsArb43B.

Meshandcollect: An Automated Multi-Crystal Data-Collection Workflow for Synchrotron Macromolecular Crystallography Beamlines.

The fully automated collection and merging of partial data sets from a series of cryocooled crystals of biological macromolecules contained on the same support is presented, as are the results of test experiments carried out on various systems.

Neuroglobin and Cytoglobin: Two New Entries in the Hemoglobin Superfamily

Neuroglobin (Ngb) and cytoglobin (Cygb) are two newly discovered intracellular members of the vertebrate hemoglobin (Hb) family. Ngb, predominantly expressed in nerve cells, is of ancient evolutionary origin and is homologous to nerve‐globins of invertebrates. Cygb, present in many different tissues, shares common ancestry with myoglobin (Mb) and can be traced to early vertebrate evolution. Ngb and Cygb display the classical three‐on‐three α‐helical globin fold and are endowed with a hexa‐coordinate heme Fe atom, in both their ferrous and ferric forms, having the heme distal HisE7 residue as the endogenous sixth ligand. Reversible intramolecular hexa‐ to penta‐coordination of the heme Fe atom modulates Ngb and Cygb ligand‐binding properties. In Ngb and Cygb, ligand migration to/from the heme distal site may be assisted by protein/matrix tunnel cavity systems. The physiological roles of Ngb and Cygb are poorly understood. Ngb may protect neuronal cells from hypoxic‐ischemic insults, may act as oxidative stress‐responsive sensor protein, and may sustain NO/O2 scavenging and/or reactive oxygen species (ROS) detoxification. Cygb, located in the cytoplasm of fibroblasts, chondroblasts, osteoblasts, and hepatic stellate cells, has been hypothesized to be involved in collagen synthesis. In neurons, Cygb, located in both cytoplasm and nucleus, may provide O2 for enzymatic reactions, and may be involved in a ROS (NO)‐signaling pathway(s). Here, we review current knowledge on Ngb and Cygb in terms of their structure, function, and evolutionary links to the well‐known human HbA and Mb.

A structured interdomain linker directs self-polymerization of human uromodulin

Significance Urinary tract infection is the most common nonepidemic bacterial infection in humans, with 150 million cases per year and a global health care cost above

Structural Basis for the Oxidation of Protein-bound Sulfur by the Sulfur Cycle Molybdohemo-Enzyme Sulfane Dehydrogenase SoxCD

6 billion. Because the urinary tract is not protected by mucus, mammals produce a molecular net that captures pathogenic bacteria in the urine and clears them from the body. By visualizing the 3D structure of its building block, glycoprotein uromodulin, we provide insights into how the net is built, and how it is compromised by mutations in patients with kidney diseases. Our work also explains nonsyndromic deafness due to mutations affecting the tectorial membrane, a similar filamentous structure in the human inner ear. Uromodulin (UMOD)/Tamm–Horsfall protein, the most abundant human urinary protein, plays a key role in chronic kidney diseases and is a promising therapeutic target for hypertension. Via its bipartite zona pellucida module (ZP-N/ZP-C), UMOD forms extracellular filaments that regulate kidney electrolyte balance and innate immunity, as well as protect against renal stones. Moreover, salt-dependent aggregation of UMOD filaments in the urine generates a soluble molecular net that captures uropathogenic bacteria and facilitates their clearance. Despite the functional importance of its homopolymers, no structural information is available on UMOD and how it self-assembles into filaments. Here, we report the crystal structures of polymerization regions of human UMOD and mouse ZP2, an essential sperm receptor protein that is structurally related to UMOD but forms heteropolymers. The structure of UMOD reveals that an extensive hydrophobic interface mediates ZP-N domain homodimerization. This arrangement is required for filament formation and is directed by an ordered ZP-N/ZP-C linker that is not observed in ZP2 but is conserved in the sequence of deafness/Crohn’s disease-associated homopolymeric glycoproteins α-tectorin (TECTA) and glycoprotein 2 (GP2). Our data provide an example of how interdomain linker plasticity can modulate the function of structurally similar multidomain proteins. Moreover, the architecture of UMOD rationalizes numerous pathogenic mutations in both UMOD and TECTA genes.

Unique thrombin inhibition mechanism by anophelin, an anticoagulant from the malaria vector.

The sulfur cycle enzyme sulfane dehydrogenase SoxCD is an essential component of the sulfur oxidation (Sox) enzyme system of Paracoccus pantotrophus. SoxCD catalyzes a six-electron oxidation reaction within the Sox cycle. SoxCD is an α2β2 heterotetrameric complex of the molybdenum cofactor-containing SoxC protein and the diheme c-type cytochrome SoxD with the heme domains D1 and D2. SoxCD1 misses the heme-2 domain D2 and is catalytically as active as SoxCD. The crystal structure of SoxCD1 was solved at 1.33 Å. The substrate of SoxCD is the outer (sulfane) sulfur of Cys-110-persulfide located at the C-terminal peptide swinging arm of SoxY of the SoxYZ carrier complex. The SoxCD1 substrate funnel toward the molybdopterin is narrow and partially shielded by side-chain residues of SoxD1. For access of the sulfane-sulfur of SoxY-Cys-110 persulfide we propose that (i) the blockage by SoxD-Arg-98 is opened via interaction with the C terminus of SoxY and (ii) the C-terminal peptide VTIGGCGG of SoxY provides interactions with the entrance path such that the cysteine-bound persulfide is optimally positioned near the molybdenum atom. The subsequent oxidation reactions of the sulfane-sulfur are initiated by the nucleophilic attack of the persulfide anion on the molybdenum atom that is, in turn, reduced. The close proximity of heme-1 to the molybdopterin allows easy acceptance of the electrons. Because SoxYZ, SoxXA, and SoxB are already structurally characterized, with SoxCD1 the structures of all key enzymes of the Sox cycle are known with atomic resolution.

Direct Evidence for a Peroxide Intermediate and a Reactive Enzyme-Substrate-Dioxygen Configuration in a Cofactor-Free Oxidase.

Anopheles mosquitoes are vectors of malaria, a potentially fatal blood disease affecting half a billion humans worldwide. These blood-feeding insects include in their antihemostatic arsenal a potent thrombin inhibitor, the flexible and cysteine-less anophelin. Here, we present a thorough structure-and-function analysis of thrombin inhibition by anophelin, including the 2.3-Å crystal structure of the human thrombin·anophelin complex. Anophelin residues 32–61 are well-defined by electron density, completely occupying the long cleft between the active site and exosite I. However, in striking contrast to substrates, the D50-R53 anophelin tetrapeptide occupies the active site cleft of the enzyme, whereas the upstream residues A35-P45 shield the regulatory exosite I, defining a unique reverse-binding mode of an inhibitor to the target proteinase. The extensive interactions established, the disruption of thrombin’s active site charge–relay system, and the insertion of residue R53 into the proteinase S1 pocket in an orientation opposed to productive substrates explain anophelin’s remarkable specificity and resistance to proteolysis by thrombin. Complementary biophysical and functional characterization of point mutants and truncated versions of anophelin unambiguously establish the molecular mechanism of action of this family of serine proteinase inhibitors (I77). These findings have implications for the design of novel antithrombotics.

Crystal structure of a junction between two Z-DNA helices.

Cofactor-free oxidases and oxygenases promote and control the reactivity of O2 with limited chemical tools at their disposal. Their mechanism of action is not completely understood and structural information is not available for any of the reaction intermediates. Near-atomic resolution crystallography supported by in crystallo Raman spectroscopy and QM/MM calculations showed unambiguously that the archetypical cofactor-free uricase catalyzes uric acid degradation via a C5(S)-(hydro)peroxide intermediate. Low X-ray doses break specifically the intermediate C5=OO(H) bond at 100 K, thus releasing O2 in situ, which is trapped above the substrate radical. The dose-dependent rate of bond rupture followed by combined crystallographic and Raman analysis indicates that ionizing radiation kick-starts both peroxide decomposition and its regeneration. Peroxidation can be explained by a mechanism in which the substrate radical recombines with superoxide transiently produced in the active site.