The 3D structure of the bacterial peptidoglycan, the major constituent of the cell wall, is one of the most important, yet still unsolved, structural problems in biochemistry. The peptidoglycan comprises alternating N-acetylglucosamine (NAG) and N-acetylmuramic disaccharide (NAM) saccharides, the latter of which has a peptide stem. Adjacent peptide stems are cross-linked by the transpeptidase enzymes of cell wall biosynthesis to provide the cell wall polymer with the structural integrity required by the bacterium. The cell wall and its biosynthetic enzymes are targets of antibiotics. The 3D structure of the cell wall has been elusive because of its complexity and the lack of pure samples. Herein we report the 3D solution structure as determined by NMR of the 2-kDa NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide) synthetic fragment of the cell wall. The glycan backbone of this peptidoglycan forms a right-handed helix with a periodicity of three for the NAG-NAM repeat (per turn of the helix). The first two amino acids of the pentapeptide adopt a limited number of conformations. Based on this structure a model for the bacterial cell wall is proposed. This pentapeptide stem participates in an interglycan cross-linking reaction, thus creating the cell wall polymer. In contrast to the two other ␤-1,4-linked glycan biopolymers, cellulose (repeating glucose) (1-4) and chitin (repeating NAG) (5-7) for which the 3D structure is solved, the structure of the bacterial cell wall has remained elusive because of its complexity and the lack of pure and discrete segments for structural study (8-18). Herein we describe the 3D structure, determined in aqueous solution by NMR, of a 2-kDa synthetic NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide) tetrasaccharide cell wall segment. The defining aspect of this structure is an ordered, right-handed helical saccharide conformation corresponding to three NAG-NAM pairs per turn of the helix. The structure of this peptidoglycan segment is the basis for a proposal for the structure of the bacterial cell wall polymer.Results and Discussion 3D Structure of the Peptidoglycan. Because of the critical significance of the cell wall to bacterial survival, and the exploitation of the cell wall biosynthetic enzymes for the chemotherapeutic intervention of infections, many experimental and theoretical studies have addressed the cell wall structure. Despite diffraction studies carried out Ͼ30 years ago on cell wall extracted from bacteria, which strongly suggested that the peptidoglycan polymer possessed regular order (11), the 3D structure of the cell wall is not known. An excellent account of the historical development of the hypotheses for the cell wall structure is given by Dmitriev, Toukach, and Ehlers in their recent review (18). The major reason for the lack of progress is the absence of a pure fragment of the cell wall, having both the peptide and disaccharide components of the peptidoglycan, for structural investigation. To address this limitation we completed the 37-step synthesis of such a segment (1...

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Background: Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol. Results: Metal binding data and crystal structures reveal that zinc inhibits pMMO at two sites. Conclusion: Zinc does not inhibit pMMO by binding at the active site but may hinder another function such as proton transfer. Significance: New insight into the function of an environmentally and industrially important enzyme has been obtained.

Friedreich's ataxia, an autosomal cardio-and neurodegenerative disorder that affects 1 in 50,000 humans, is caused by decreased levels of the protein frataxin. Although nuclear encoded, frataxin is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting monomeric frataxin might function as the common iron donor.In order to provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron loaded monomer and the protein can bind 2 ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal binding sites is consistent with a 6 coordinate iron-(oxygen and nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. Based on our results, we have developed a model for how we believe yeast frataxin interacts with iron. Keywords Briefs:We present a characterization of monomeric yeast frataxin's iron binding ability. Various spectroscopic techniques were applied to help characterize the iron binding affinity of yeast frataxin, the oligomeric state of the protein, specific amino acids affected by the presence of iron and finally the metal-ligand coordination geometry. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 August 19. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript patients, is a direct result of a trinucleotide repeat expansion in the first intron of the gene for the protein frataxin; this expansion disrupts gene transcription leading to a frataxin deficiency in humans (4). The inability to produce frataxin is associated with mitochondrial accumulation of iron that is biologically unavailable. In humans, the breakdown in normal iron regulation pathways resulting from frataxin deficiency is exemplified physiologically by the degeneration of sensory neurons in the dorsal root ganglia, hypertrophic cardiomyopathy and diabetes mellitus (5). In yeast, reintroduction of frataxin into knockout cells results in restored bioavailability of the accumulated metal (6). These data indicate frataxin is required for retaining bi...

After budding, the human immunodeficiency virus (HIV) must 'mature' into an infectious viral particle. Viral maturation requires proteolytic processing of the Gag polyprotein at the matrix-capsid junction, which liberates the capsid (CA) domain to condense from the spherical protein coat of the immature virus into the conical core of the mature virus. We propose that upon proteolysis, the amino-terminal end of the capsid refolds into a β-hairpin/helix structure that is stabilized by formation of a salt bridge between the processed amino-terminus (Pro1) and a highly conserved aspartate residue (Asp51). The refolded amino-terminus then creates a new CA-CA interface that is essential for assembling the condensed conical core. Consistent with this model, we found that recombinant capsid proteins with as few as four matrix residues fused to their aminotermini formed spheres in vitro, but that removing these residues refolded the capsid amino-terminus and redirected protein assembly from spheres to cylinders. Moreover, point mutations throughout the putative CA-CA interface blocked capsid assembly in vitro, core assembly in vivo and viral infectivity. Disruption of the conserved amino-terminal capsid salt bridge also abolished the infectivity of Moloney murine leukemia viral particles, suggesting that lenti-and oncoviruses mature via analogous pathways.