Simone Zuccotti

Crystal Structure and Anion Binding in the Prokaryotic Hydrogenase Maturation Factor HypF Acylphosphatase-like Domain

[NiFe]-hydrogenases require a set of complementary and regulatory proteins for correct folding and maturation processes. One of the essential regulatory proteins, HypF (82kDa) contains a N-terminal acylphosphatase (ACT)-like domain, a sequence motif shared with enzymes catalyzing O-carbamoylation, and two zinc finger motifs similar to those found in the DnaJ chaperone. The HypF acylphosphatase domain is thought to support the conversion of carbamoylphosphate into CO and CN(-), promoting coordination of these ligands to the hydrogenase metal cluster. It has been shown recently that the HypF N-terminal domain can aggregate in vitro to yield fibrils matching those formed by proteins linked to amyloid diseases. The 1.27A resolution HypF acylphosphatase domain crystal structure (residues 1-91; R-factor 13.1%) shows a domain fold of betaalphabetabetaalphabeta topology, as observed in mammalian acylphosphatases specifically catalyzing the hydrolysis of the carboxyl-phosphate bonds in acylphosphates. The HypF N-terminal domain can be assigned to the ferredoxin structural superfamily, to which RNA-binding domains of small nuclear ribonucleoproteins and some metallochaperone proteins belong. Additionally, the HypF N-terminal domain displays an intriguing structural relationship to the recently discovered ACT domains. The structures of different HypF acylphosphatase domain complexes show a phosphate binding cradle comparable to the P-loop observed in unrelated phosphatase families. On the basis of the catalytic mechanism proposed for acylphosphatases, whereby residues Arg23 and Asn41 would support substrate orientation and the nucleophilic attack of a water molecule on the phosphate group, fine structural features of the HypF N-terminal domain putative active site region may account for the lack of acylphosphatase activity observed for the expressed domain. The crystallographic analyses here reported were undertaken to shed light on the molecular bases of inactivity, folding, misfolding and aggregation of the HypF N-terminal acylphosphatase domain.

Structure, conformational stability, and enzymatic properties of acylphosphatase from the hyperthermophile Sulfolobus solfataricus.

The structure of AcP from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined by 1H‐NMR spectroscopy and X‐ray crystallography. Solution and crystal structures (1.27 Å resolution, R‐factor 13.7%) were obtained on the full‐length protein and on an N‐truncated form lacking the first 12 residues, respectively. The overall Sso AcP fold, starting at residue 13, displays the same βαββαβ topology previously described for other members of the AcP family from mesophilic sources. The unstructured N‐terminal tail may be crucial for the unusual aggregation mechanism of Sso AcP previously reported. Sso AcP catalytic activity is reduced at room temperature but rises at its working temperature to values comparable to those displayed by its mesophilic counterparts at 25–37°C. Such a reduced activity can result from protein rigidity and from the active site stiffening due the presence of a salt bridge between the C‐terminal carboxylate and the active site arginine. Sso AcP is characterized by a melting temperature, Tm, of 100.8°C and an unfolding free energy, ΔG  U‐FH 2O , at 28°C and 81°C of 48.7 and 20.6 kJ mol−1, respectively. The kinetic and structural data indicate that mesophilic and hyperthermophilic AcPs display similar enzymatic activities and conformational stabilities at their working conditions. Structural analysis of the factor responsible for Sso AcP thermostability with respect to mesophilic AcPs revealed the importance of a ion pair network stabilizing particularly the β‐sheet and the loop connecting the fourth and fifth strands, together with increased density packing, loop shortening and a higher α‐helical propensity. Proteins 2006.

Engineering Peroxidase Activity in Myoglobin: The Haem Cavity Structure and Peroxide Activation in the T67R/S92D Mutant and its Derivative Reconstituted with Protohaemin-L-Histidine.

Atomic co-ordinates and structure factors for the T67R/S92D metMbCN mutant have been deposited with the Protein Data Bank, under accession codes 1h1x and r1h1xsf, respectively. Protein engineering and cofactor replacement have been employed as tools to introduce/modulate peroxidase activity in sperm whale Mb (myoglobin). Based on the rationale that haem peroxidase active sites are characterized by specific charged residues, the Mb haem crevice has been modified to host a haem-distalpropionate Arg residue and a proximal Asp, yielding the T67R/S92D Mb mutant. To code extra conformational mobility around the haem, and to increase the peroxidase catalytic efficiency, the T67R/S92D Mb mutant has been subsequently reconstituted with protohaem-L-histidine methyl ester, yielding a stable derivative, T67R/S92D Mb-H. The crystal structure of T67R/S92D cyano-metMb (1.4 A resolution; R factor, 0.12) highlights a regular haem-cyanide binding mode, and the role for the mutated residues in affecting the haem propionates as well as the neighbouring water structure. The conformational disorder of the haem propionate-7 is evidenced by the NMR spectrum of the mutant. Ligand-binding studies show that the iron(III) centres of T67R/S92D Mb, and especially of T67R/S92D Mb-H, exhibit higher affinity for azide and imidazole than wild-type Mb. In addition, both protein derivatives react faster than wild-type Mb with hydrogen peroxide, showing higher peroxidase-like activity towards phenolic substrates. The catalytic efficiency of T67R/S92D Mb-H in these reactions is the highest so far reported for Mb derivatives. A model for the protein-substrate interaction is deduced based on the crystal structure and on the NMR spectra of protein-phenol complexes.

Three-dimensional structural characterization of a novel Drosophila melanogaster acylphosphatase.

Analysis of the Drosophila melanogaster EST database led to the discovery and cloning of a novel acylphosphatase. The CG18505 gene coding for a new enzyme (AcPDro2) is clearly distinct from the previously described CG16870Acyp gene, which also codes for a D. melanogaster acylphosphatase (AcPDro). The putative catalytic residues, together with residues held to stabilize the acylphosphatase fold, are conserved in the two encoded proteins. Crystals of AcPDro2, which belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 45.8, c = 98.6 angstroms, gamma = 120 degrees, allowed the solution of the protein structure by molecular replacement and its refinement to 1.5 angstroms resolution. The AcPDro2 active-site structure is discussed.

Preliminary crystallographic characterization of the human β2 microglobulin His31Tyr mutant in a tetrameric assembly

Patients receiving prolonged haemodialysis treatment are exposed to a variety of arthropathies and bone lesions arising from deposition of amyloid material in the skeletal system. beta2 microglobulin is the 11.7 kDa light chain of the class I major histocompatibility complex, from which it is normally released to plasmatic fluids, transported to kidneys and excreted. Owing to renal failure it accumulates, giving rise to dialysis-related amyloidosis, a severe disease found in patients receiving dialysis for several years. The three-dimensional structure of beta2 microglobulin is known to be based on a seven-stranded beta-sandwich fold, typical of the class C immunoglobulin superfamily. Analysis of the protein fold in different mutants and/or crystal environments and of its structural stability may help in understanding the molecular bases of amyloid fibril formation and of diseases related to protein misfolding. Here, the preliminary crystallographic analysis of the His31Tyr beta2 microglobulin mutant, designed to abolish the copper-ion binding observed in the wild-type protein, is presented. The protein mutant displays increased fold stability, faster folding kinetics and crystallizes in the tetragonal C222(1) space group, with unit-cell parameters a = 105.2, b = 150.2, c = 93.7 A and four molecules per asymmetric unit.

Crystallization and preliminary X-ray characterization of the acylphosphatase-like domain from the Escherichia coli hydrogenase maturation factor HypF

Maturation of prokaryotic hydrogenase involves several protein factors, among which is the accessory protein HypF, which hosts the consensus sequence of acylphosphatases and a sequence motif common to proteins catalyzing O-carbamoylations. The specific functions of HypF are largely unknown, although it has been observed that CN(-) and CO ligands at the hydrogenase Ni,Fe active centre originate from carbamoylphosphate. The HypF N-terminal domain (91 residues, acylphosphatase-like domain) has been crystallized in two different crystal forms belonging to the orthorhombic P2(1)2(1)2(1) space group (unit-cell parameters a = 35.5, b = 59.8, c = 87.6 A) and to the rhombohedral space group R32 (unit-cell parameters a = b = 58.1, c = 155.6 A in the hexagonal setting).

Preliminary characterization of two different crystal forms of acylphosphatase from the hyperthermophile archaeon Sulfolobus solfataricus

Acylphosphatase is a ubiquitous small enzyme that was first characterized in mammals. It is involved in the hydrolysis of carboxyl-phosphate bonds in several acylphosphate substrates, such as carbamoylphosphate and 1,3-biphosphoglycerate; however, a consensus on acylphosphatase action in vivo has not yet been reached. Recent investigations have focused on acylphosphatases from lower phyla, such as Drosophila melanogaster and Escherichia coli, in view of the application of these small proteins as models in the study of folding, misfolding and aggregation processes. An acylphosphatase from the hyperthermophilic archaeon Sulfolobus solfataricus has been cloned, expressed and purified. Here, the growth and characterization of a triclinic and a monoclinic crystal form of the hyperthermophilic enzyme are reported; X-ray diffraction data have been collected to 1.27 and 1.90 A resolution, respectively.