Domenico Bordo

CD81 extracellular domain 3D structure: insight into the tetraspanin superfamily structural motifs

Human CD81, a known receptor for hepatitis C virus envelope E2 glycoprotein, is a transmembrane protein belonging to the tetraspanin family. The crystal structure of human CD81 large extracellular domain is reported here at 1.6 Å resolution. Each subunit within the homodimeric protein displays a mushroom‐like structure, composed of five α‐helices arranged in ‘stalk’ and ‘head’ subdomains. Residues known to be involved in virus binding can be mapped onto the head subdomain, providing a basis for the design of antiviral drugs and vaccines. Sequence analysis of 160 tetraspanins indicates that key structural features and the new protein fold observed in the CD81 large extracellular domain are conserved within the family. On these bases, it is proposed that tetraspanins may assemble at the cell surface into homo‐ and/or hetero‐dimers through a conserved hydrophobic interface located in the stalk subdomain, while interacting with other liganding proteins, including hepatitis C virus E2, through the head subdomain. The topology of such interactions provides a rationale for the assembly of the so‐called tetraspan‐web.

The Three-Dimensional Structure of the Human NK Cell Receptor NKp44, a Triggering Partner in Natural Cytotoxicity

Natural killer (NK) cells direct cytotoxicity against tumor or virally infected cells. NK cell activation depends on a fine balance between inhibitory and activating receptors. NKp44 is a cytotoxicity activating receptor composed of one Ig-like extracellular domain, a transmembrane segment, and a cytoplasmic domain. The 2.2 A crystal structure shows that the NKp44 Ig domain forms a saddle-shaped dimer, where a charged surface groove protrudes from the core structure in each subunit. NKp44 Ig domain disulfide bridge topology defines a new Ig structural subfamily. The data presented are a first step toward understanding the molecular basis for ligand recognition by natural cytotoxicity receptors, whose key role in the immune system is established, but whose cellular ligands are still elusive.

Mutational analysis of the ACVR1 gene in Italian patients affected with fibrodysplasia ossificans progressiva: confirmations and advancements

Fibrodysplasia ossificans progressiva (FOP, MIM 135100) is a rare genetic disorder characterized by congenital great toe malformations and progressive heterotopic ossification transforming skeletal muscles and connective tissues to bone following a well-defined anatomic pattern of progression. Recently, FOP has been associated with a specific mutation of ACVR1, the gene coding for a bone morphogenetic protein type I receptor. The identification of ACVR1 as the causative gene for FOP now allows the genetic screening of FOP patients to identify the frequency of the identified recurrent ACVR1 mutation and to investigate genetic variability that may be associated with this severely debilitating disease. We report the screening for mutations in the ACVR1 gene carried out in a cohort of 17 Italian patients. Fifteen of these displayed the previously described c.617G>A mutation, leading to the R206H substitution in the GS domain of the ACVR1 receptor. In two patients, we found a novel mutation c.774G>C, leading to the R258S substitution in the kinase domain of the ACVR1 receptor. In the three-dimensional model of protein structure, R258 maps in close proximity to the GS domain, a key regulator of ACVR1 activity, where R206 is located. The GS domain is known to bind the regulatory protein FKBP12 and to undergo multiple phosphorylation events that trigger a signaling cascade inside the cell. The novel amino-acid substitution is predicted to influence either the conformation/stability of the GS region or the binding affinity with FKBP12, resulting in a less stringent inhibitory control on the ACVR1 kinase activity.

Re-Evaluation of Amino Acid Sequence and Structural Consensus Rules for Cysteine-Nitric Oxide Reactivity

Abstract Nitric oxide (NO), produced in different cell types through the conversion of Larginine into Lcitrulline by the enzyme NO synthase, has been proposed to exert its action in several physiological and pathological events. The great propensity for nitrosothiol formation and breakdown represents a mechanism which modulates the action of macromolecules containing NOreactive Cys residues at their active centre and/or allosteric sites. Based on the human haemoglobin (Hb) structure and accounting for the known acidbase catalysed Cys?93-nitrosylation and Cys?93NOdenitrosylation processes, the putative amino acid sequence (Lys/Arg/His/Asp/Glu)Cys(Asp/Glu) (sites 1, 0, and + 1, respectively) has been proposed as the minimum consensus motif for CysNO reactivity. Although not found in human Hb, the presence of a polar amino acid residue (Gly/Ser/Thr/Cys/Tyr/Asn/Gln) at the 2 position has been observed in some NOreactive protein sequences (e.g., NMDA receptors). However, the most important component of the tri or tetrapeptide consensus motif has been recognised as the Cys(Asp/Glu) pair [Stamler et al., Neuron (1997) 18, 691 696]. Here, we analyse the threedimensional structure of several proteins containing NOreactive Cys residues, and show that their nitrosylation and denitrosylation processes may depend on the CysS? atomic structural microenvironment rather than on the tri or tetrapeptide sequence consensus motif.

Evolution of protein cores: Constraints in point mutations as observed in globin tertiary structures

The amino acid sequences of ten globin chain tertiary structures were aligned and structurally equivalenced by spatial superposition of main-chain C alpha atoms. A search was then performed for structurally equivalent residue pairs that were buried in the protein core and that had mutated but maintained similar unmutated environments. Residues with atoms in contact with such central residue pairs define their environments. Such examples of point mutations would represent in vivo site-directed mutagenesis as would be observed in evolution. A search for mutated but exposed equivalent central residues was also performed. The constraints placed on the characteristics of the mutated residues (e.g., side-chain volume, polarity, radius of gyration) allow suggestions for the evolutionary modes of protein core and surface development as well as residue substitution guidelines to maintain structural stability in protein engineering and design.

Escherichia coli GlpE Is a Prototype Sulfurtransferase for the Single-Domain Rhodanese Homology Superfamily

BACKGROUND Rhodanese domains are structural modules occurring in the three major evolutionary phyla. They are found as single-domain proteins, as tandemly repeated modules in which the C-terminal domain only bears the properly structured active site, or as members of multidomain proteins. Although in vitro assays show sulfurtransferase or phosphatase activity associated with rhodanese or rhodanese-like domains, specific biological roles for most members of this homology superfamily have not been established. RESULTS Eight ORFs coding for proteins consisting of (or containing) a rhodanese domain bearing the potentially catalytic Cys have been identified in the Escherichia coli K-12 genome. One of these codes for the 12-kDa protein GlpE, a member of the sn-glycerol 3-phosphate (glp) regulon. The crystal structure of GlpE, reported here at 1.06 A resolution, displays alpha/beta topology based on five beta strands and five alpha helices. The GlpE catalytic Cys residue is persulfurated and enclosed in a structurally conserved 5-residue loop in a region of positive electrostatic field. CONCLUSIONS Relative to the two-domain rhodanese enzymes of known three-dimensional structure, GlpE displays substantial shortening of loops connecting alpha helices and beta sheets, resulting in radical conformational changes surrounding the active site. As a consequence, GlpE is structurally more similar to Cdc25 phosphatases than to bovine or Azotobacter vinelandii rhodaneses. Sequence searches through completed genomes indicate that GlpE can be considered to be the prototype structure for the ubiquitous single-domain rhodanese module.

A persulfurated cysteine promotes active site reactivity in Azotobacter vinelandii rhodanese

Abstract Active site reactivity and specificity of RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) from Azotobacter vinelandii, have been investigated through ligand binding, sitedirected mutagenesis, and Xray crystallographic techniques, in a combined approach. In native RhdA the active site Cys230 is found persulfurated; fluorescence and sulfurtransferase activity measurements show that phosphate anions interact with Cys230 persulfide sulfur atom and modulate activity. Crystallographic analyses confirm that phosphate and hypophosphite anions react with native RhdA, removing the persulfide sulfur atom from the active site pocket. Considering that RhdA and the catalytic subunit of Cdc25 phosphatases share a common threedimensional fold as well as active site Cys (catalytic) and Arg residues, two RhdA mutants carrying a single amino acid insertion at the active site loop were designed and their phosphatase activity tested. The crystallographic and functional results reported here show that specific sulfurtransferase or phosphatase activities are strictly related to precise tailoring of the catalytic loop structure in RhdA and Cdc25 phosphatase, respectively.

A SOD1 gene mutation in a patient with slowly progressing familial ALS

Article abstract We report a new missense mutation (Gly12Ala) in exon 1 of the Cu/Zn superoxide dismutase (SOD1) gene in a 67-year-old patient with familial ALS (FALS). The clinical course showed an unusually slow progression. The enzymatic activity of the mutated SOD1 was 80% of normal. At the molecular level, the Gly12Ala mutation occurs in a region outside the active site and may lead to local distortion strain in the protein structure.

Patterns in ionizable side chain interactions in protein structures

In a selected set of 44 high‐resolution, non‐homologous protein structures, the intramolecular hydrogen bonds or salt bridges formed by ionizable amino acid side chains were identified and analyzed. The analysis was based on the investigation of several properties of the involved residues such as their solvent exposure, their belonging to a certain secondary structural element, and their position relative to the N‐ and C‐termini of their respective structural element. It was observed that two‐thirds of the interactions made by basic or acidic side chains are hydrogen bonds to polar uncharged groups. In particular, the majority (78%) of the hydrogen bonds between ionizable side chains and main chain polar groups (sch:mch bonds) involved at least one buried atom, and in 42% of the cases both interacting atoms were buried. In α‐helices, the sch:mch bonds observed in the proximity of the C‐ and N‐termini show a clear preference for acidic and basic side chains, respectively. This appears to be due to the partial charges of peptide group atoms at the termini of α‐helices, which establish energetically favorable electrostatic interactions with side chain carrying opposite charge, at distances even greater than 4.5 Å. The sch:mch interactions involving ionizable side chains that belong either to β‐strands or to the central part of α‐helices are based almost exclusively on basic residues. This results from the presence of main chain carbonyl oxygen atoms in the protein core which have unsatisfied hydrogen bonding capabilities.

Structural characterization of the As/Sb reductase LmACR2 from Leishmania major.

The arsenate/antimonate reductase LmACR2 has been recently identified in the genome of Leishmania major. Besides displaying phosphatase activity in vitro, this enzyme is able to reduce both As(V) and Sb(V) to their respective trivalent forms and is involved in the activation of Pentostan, a drug containing Sb(V) used in the treatment of leishmaniasis. LmACR2 displays sequence and functional similarity with the arsenate reductase ScACR2 from Saccharomyces cerevisiae, and both proteins are homologous to the catalytic domain of Cdc25 phosphatases, which, in turn, belong to the rhodanese/Cdc25 phosphatase superfamily. In this work, the three-dimensional structure of LmACR2 has been determined with crystallographic methods and refined at 2.15 A resolution. The protein structure maintains the overall rhodanese fold, but substantial modifications are observed in secondary structure position and length. However, the conformation of the active-site loop and the position of the catalytic residue Cys75 are unchanged with respect to the Cdc25 phosphatases. From an evolutionary viewpoint, LmACR2 and the related arsenate reductases form, together with the known Cdc25 phosphatases, a well-defined subfamily of the rhodanese/Cdc25 phosphatase superfamily, characterized by a 7-amino-acid-long active-site loop that is able to selectively bind substrates containing phosphorous, arsenic, or antinomy. The evolutionary tree obtained for these proteins shows that, besides the active-site motif CE[F/Y]SXXR that characterizes Cdc25 phosphatase, the novel CALSQ[Q/V]R motif is also conserved in sequences from fungi and plants. Similar to Cdc25 phosphatase, these proteins are likely involved in cell cycle control. The active-site composition of LmACR2 (CAQSLVR) does not belong to either group, but gives to the enzyme a bifunctional activity of both phosphatase and As/Sb reductase. The subtle dependence of substrate specificity on the amino acid composition of the active-site loop displays the versatility of the ubiquitous rhodanese domain.