Structural and functional characterization of Streptomyces plicatus beta-N-acetylhexosaminidase by comparative molecular modeling and site-directed mutagenesis

We have sequenced the Streptomyces plicatus beta-N-acetylhexosaminidase (SpHex) gene and identified the encoded protein as a member of family 20 glycosyl hydrolases. This family includes human beta-N-acetylhexosaminidases whose deficiency results in various forms of GM2 gangliosidosis. Based upon the x-ray structure of Serratia marcescens chitobiase (SmChb), we generated a three-dimensional model of SpHex by comparative molecular modeling. The overall structure of the enzyme is very similar to homology modeling-derived structures of human beta-N-acetylhexosaminidases, with differences being confined mainly to loop regions. From previous studies of the human enzymes, sequence alignments of family 20 enzymes, and analysis of the SmChb x-ray structure, we selected and mutated putative SpHex active site residues. Arg162 --> His mutation increased Km 40-fold and reduced Vmax 5-fold, providing the first biochemical evidence for this conserved Arg residue (Arg178 in human beta-N-acetylhexosaminidase A (HexA) and Arg349 in SmChb) as a substrate-binding residue in a family 20 enzyme, a finding consistent with our three-dimensional model of SpHex. Glu314 --> Gln reduced Vmax 296-fold, reduced Km 7-fold, and altered the pH profile, consistent with it being the catalytic acid residue as suggested by our model and other studies. Asp246 --> Asn reduced Vmax 2-fold and increased Km only 1.2-fold, suggesting that Asp246 may play a lesser role in the catalytic mechanism of this enzyme. Taken together with the x-ray structure of SmChb, these studies suggest a common catalytic mechanism for family 20 glycosyl hydrolases.     

Structural characterization of four genetic variants of human serum albumin associated with alloalbuminemia in Italy

A long-term electrophoretic survey on plasma proteins, which was carried out in several clinical laboratories in Italy, identified 28 different genetic variants of human serum albumin and four cases of analbuminemia. We have previously characterized 16 point mutations, 3 C-terminal mutants, and the genetic defects in two analbuminemic subjects. Here, we report the molecular defects of four alloalbumins that have been characterized by protein structural analysis. Of these, three represent new single-point mutations: albumins Tregasio, Val122-->Glu, Bergamo, Asp314-->Gly, and Maddaloni, Val533-->Met. The fourth, albumin Besana Brianza, has the same Asp494-->Asn mutation that introduces a glycosylation site which has been previously reported in a variant from New Zealand, albumin Casebrook. However, in contrast to albumin Casebrook, albumin Besana Brianza is only partially glycosylated and the oligosaccharide is heterogeneous, consisting of a biantennary complex type N-glycan with either two or one sialic acid residue(s) on the antennae. Both albumin Maddaloni and Besana Brianza represent mutations at hypermutable CpG dinucleotide sites; albumin Maddaloni is a mutant that does not involve a charged amino acid.     

Reversible peptide folding in solution by molecular dynamics simulation

Long-standing questions on how peptides fold are addressed by the simulation at different temperatures of the reversible folding of a peptide in solution in atomic detail. Molecular dynamics simulations correctly predict the structure that is thermodynamically stable at 298 K, irrespective of the initial peptide conformation. The rate of folding and the free energy of folding at different temperatures are estimated. Although the conformational space potentially accessible to the peptide is extremely large, very few conformers (10(1) to 10(2)) are significantly populated at 20 K above the melting temperature. This implies that the search problem in peptide (or even protein) folding is surmountable using dynamics simulations.     

Antileukoprotease in human skin: an antibiotic peptide constitutively produced by keratinocytes

Antileukoprotease (ALP), also known as mucous protease inhibitor or secretory leukoprotease inhibitor, resembles one of the major antiproteases present in human body fluids. It is capable of preventing proteolytic degradation of extracellular matrix proteins by neutrophil-derived serine proteases. ALP was isolated from human callus and detected in supernatants of cultured human primary keratinocytes. ALP mRNA was constitutively expressed in keratinocytes and the expression was not significantly affected by TNF alpha or Interferon gamma stimulation. In microbicidal assays recombinant ALP exhibited antimicrobial activity against several human skin associated microorganisms like P. aeruginosa, S. aureus, S. epidermidis, and C. albicans, indicating that ALP may actively participate in mechanisms allowing homeostasis of bacterial and yeast colonization on human skin. Thus, ALP represents a major soluble serine protease inhibitor and antimicrobial agent expressed in human skin and seems to contribute to the high resistance of the epidermis against proteolysis and infections.     

Structural characterization of an abnormally cross-linked muropeptide dimer that is accumulated in the peptidoglycan of methicillin- and cefotaxime-resistant mutants of Staphylococcus aureus

Laboratory mutants of Staphylococcus aureus strain ATCC 8325 (27S) selected for increased minimal inhibitory concentration (MIC) values to methicillin and cefotaxime showed increased rates of cell wall turnover and detergent-induced autolysis in virtual parallel with the increasing MIC for the antibiotic. Also in parallel with the increasing MICs for the particular antibiotic used in the selection was the gradual accumulation of an unusual muropeptide in the peptidoglycan of the mutants, muropeptide 12, which is a minor component of the cell wall of the parental strain. Analysis of muropeptide 12, its peptide derivative, and its lysostaphin degradation products by high pressure liquid chromatography, Edman degradation, and mass spectrometry suggests that muropeptide 12 is a dimer in which the two monomeric components are interlinked by two pentaglycyl cross-bridges, thus generating a 14-member macrocyclic ring structure. This unusual cross-linked structure may be the product of the abnormal activity of penicillin-binding protein 2 which has grossly reduced antibiotic binding capacity in the mutant staphylococci.     

Solution conformation of the Pseudomonas syringae MSU 16H phytotoxic lipodepsipeptide Pseudomycin A determined by computer simulations using distance geometry and molecular dynamics from NMR data

Pseudomycin A is a cyclic lipodepsinonapeptide phytotoxin produced by a strain of the plant pathogenic bacterium Pseudomonas syringae. Like other members of this family of bacterial metabolites, it is characterised by a fatty acylated cyclic peptide with mixed chirality and lactonic closure. Several biological activities of Pseudomycin A are lower than those found for some of its congeners, a difference which might depend on the diverse number and distribution of charged residues in the peptide moiety. Hence, it was of interest to investigate its conformation in solution. After the complete interpretation of the two-dimensional NMR spectra, NOE data were obtained and the structure was determined by computer simulations, applying distance geometry and molecular dynamics procedures. The conformation of the large ring of Pseudomycin A in solution includes three rigid structural regions interrupted by three short flexible regions that act as hinges. The overall three-dimensional structure of the cyclic moiety is similar to that of previously studied bioactive lipodepsinonapeptides produced by other pseudomonads.     

Structural analysis of substrate binding by the molecular chaperone DnaK

DnaK and other members of the 70-kilodalton heat-shock protein (hsp70) family promote protein folding, interaction, and translocation, both constitutively and in response to stress, by binding to unfolded polypeptide segments. These proteins have two functional units: a substrate-binding portion binds the polypeptide, and an adenosine triphosphatase portion facilitates substrate exchange. The crystal structure of a peptide complex with the substrate-binding unit of DnaK has now been determined at 2.0 angstroms resolution. The structure consists of a beta-sandwich subdomain followed by alpha-helical segments. The peptide is bound to DnaK in an extended conformation through a channel defined by loops from the beta sandwich. An alpha-helical domain stabilizes the complex, but does not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, which suggests a model of conformation-dependent substrate binding that features a latch mechanism for maintaining long lifetime complexes.     

Intrinsic nature of the three-dimensional structure of proteins as determined by distance geometry with good sampling properties

A protocol for distance geometry calculation is shown to have excellent sampling properties in the determination of three-dimensional structures of proteins from nuclear magnetic resonance (NMR) data. This protocol uses a simulated annealing optimization employing mass-weighted molecular dynamics in four-dimensional space (Havel, T.F. (1991) Prog. Biophys. Mol. Biol., 56, 43-78). It attains an extremely large radius of convergence, allowing a random coil conformation to be used as the initial estimate for the succeeding optimization process. Computations are performed with four systems of simulated distance data as tests of the protocol, using an unconstrained L-alanine 30mer and three different types of proteins, bovine pancreatic trypsin inhibitor, the alpha-amylase inhibitor Tendamistat, and the N-terminal domain of the 434-repressor. The test of the unconstrained polypeptide confirms that the sampled conformational space is that of the statistical random coil. In the larger and more complicated systems of the three proteins, the protocol gives complete convergence of the optimization without any trace of initial structure dependence. As a result of an exhaustive conformational sampling by the protocol, the intrinsic nature of the structures generated with distance restraints derived from NMR data has been revealed. When the sampled structures are compared with the corresponding X-ray structures, we find that the averages of the sampled structures always show a certain pattern of discrepancy from the X-ray structure. This discrepancy is due to the short distance nature of the distance restraints, and correlates with the characteristic shape of the protein molecule.     

Structural organization in peptide fragments of cytochrome c by heme binding

Prosthetic groups are often important structural organizers of proteins as well as essential functional components. Insertion of prosthetic groups is usually spontaneous, and implies an apoprotein that is partially preorganized to provide a recognition surface for specific binding. Cytochrome c is distinguished by having its heme attached by a dedicated heme lyase through thioether links to cysteine side-chains, and the apoprotein shows no evidence of preorganization under physiological conditions. Nevertheless, addition of heme to two short fragments of cytochrome c enhances helical structure substantially (from approximately 8% to approximately 22%), an effect that depends on iron ligation but not thioether linkage. The helical segments in the corresponding parts of the native holoprotein have little contact surface with heme, implying that the increased helical structure in the fragment complex may depend on tertiary interactions. The absence of the intervening polypeptide chain suggests that the complex represents a relatively independent folded subdomain.     

Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy

Magainin 2 is a 23-residue peptide that forms an amphipathic alpha-helix in membrane environments. It functions as an antibiotic and is known to disrupt the electrochemical gradients across the cell membranes of many bacteria, fungi, and some tumor cells, although it does not lyse red blood cells. One- and two-dimensional solid-state 15N NMR spectra of specifically 15N-labeled magainin 2 in oriented bilayer samples show that the secondary structure of essentially the entire peptide is alpha-helix, immobilized by its interactions with the phospholipids, and oriented parallel to the membrane surface.     

Measurement of the degree of coupled isotopic enrichment of different positions in an antibiotic peptide by NMR

An experimental strategy for determining the extent to which multiply isotopically labeled fragments are incorporated intact into relatively complicated compounds of interest is presented. The NMR methods employed are based on isotope-filtered one-dimensional spectra and difference HSQC spectra incorporating a spin echo designed to report on the presence of a second NMR active isotope at a coupled site. They supplement existing methods for determining the extent of isotopic incorporation at individual sites to reveal whether two coupled labeled sites in a precursor are incorporated as an intact unit into products. The methods described also circumvent 1H signal overlap and distinguish between the effects of different nitrogens coupled to individual carbons. The somewhat complicated case of valclavam illustrates the method's utility in measuring the J coupling constants between 13C and nearby sites that are only fractionally labeled with 15N, and measuring the fraction of molecules in which 13C is coupled to 15N, at each of several sites. The 15N of [2-13C, 15N]-labeled glycine is found to be incorporated into all three N positions of valclavam but most heavily into the N11 position. Specifically, 15N and 13C are incorporated into the N11 and C10 positions together as an 15N13C fragment approximately 8% of the time, whereas 15N is incorporated largely independently at the other positions.