No abstract

The N-terminal domain of HIV-1 glycoprotein 41000 (FP; residues 1--23; AVGIGALFLGFLGAAGSTMGARSCONH(2)) participates in fusion processes underlying virus--cell infection. Here, we use physical techniques to study the secondary conformation of synthetic FP in aqueous, structure-promoting, lipid and biomembrane environments. Circular dichroism and conventional, (12)C-Fourier transform infrared (FTIR) spectroscopy indicated the following alpha-helical levels for FP in 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) liposomes-hexafluoroisopropanol (HFIP)>trifluoroethanol (TFE)>phosphate-buffered saline (PBS). (12)C-FTIR spectra also showed disordered FP structures in these environments, along with substantial beta-structures for FP in TFE or PBS. In further experiments designed to map secondary conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using a suite of FP peptides labeled with (13)C-carbonyl at multiple sites. Combining these (13)C-enhanced FTIR results with molecular simulations indicated the following model for FP in HFIP: alpha-helix (residues 3-16) and random and beta-structures (residues 1-2 and residues 17-23). Additional (13)C-FTIR analysis indicated a similar conformation for FP in POPG at low peptide loading, except that the alpha-helix extends over residues 1-16. At low peptide loading in either human erythrocyte ghosts or lipid extracts from ghosts, (13)C-FTIR spectroscopy showed alpha-helical conformations for the central core of FP (residues 5-15); on the other hand, at high peptide loading in ghosts or lipid extracts, the central core of FP assumed an antiparallel beta-structure. FP at low loading in ghosts probably inserts deeply as an alpha-helix into the hydrophobic membrane bilayer, while at higher loading FP primarily associates with ghosts as an aqueous-accessible, beta-sheet. In future studies, (13)C-FTIR spectroscopy may yield residue-specific conformations for other membrane-bound proteins or peptides, which have been difficult to analyze with more standard methodologies.

The pathogen Staphylococcus aureus uses iron-regulated surface determinant (Isd) proteins to scavenge the essential nutrient iron from host hemoproteins. . High affinity binding to these structurally unrelated proteins requires residues located within a conserved aromatic motif that is positioned at the end of the ␤-barrel structure. Interestingly, this site is quite malleable, as other NEAT domains use it to bind heme. We also demonstrate that the IsdC NEAT domain can capture heme directly from Hb, suggesting that there are multiple pathways for heme transfer across the cell wall.

Many surface proteins are anchored to the cell wall by the action of sortase enzymes, a recently discovered family of cysteine transpeptidases. As the surface proteins of human pathogens are frequently required for virulence, the sortase-mediated anchoring reaction represents a potential target for new anti-infective agents. It has been suggested that the sortase from Staphylococcus aureus (SrtA), may use a similar catalytic strategy as the papain cysteine proteases, holding its Cys 184 side chain in an active configuration through a thiolate-imidazolium ion interaction with residue His 120 . To investigate the mechanism of transpeptidation, we have synthesized a peptidyl-vinyl sulfone substrate mimic that irreversibly inhibits SrtA. Through the study of the pH dependence of SrtA inhibition and NMR, we have estimated the pK a s of the active site thiol (Cys 184 ) and imidazole (His 120 ) to be ϳ9.4 and 7.0, respectively. These measurements are inconsistent with the existence of a thiolate-imidazolium ion pair and suggest a general base catalysis mechanism during transpeptidation.

Staphylococcus aureus scavenges heme-iron from host hemoproteins using iron-regulated surface determinant (Isd) proteins. IsdC is the central conduit through which heme is passed across the cell wall and binds this molecule using a NEAr Transporter (NEAT) domain. NMR spectroscopy was used to determine the structure of IsdC in complex with a heme analog, zinc-substituted protoporphyrin IX (ZnPPIX). The backbone coordinates of the ensemble of conformers representing the structure exhibit a root mean square deviation to the mean structure of 0.53 ؎ 0.11 Å . IsdC partially buries protoporphyrin within a large hydrophobic pocket that is located at the end of its ␤-barrel structure. The central metal ion of the analog adopts a pentacoordinate geometry in which a highly conserved tyrosine residue serves as a proximal ligand. Consistent with the structure and its role in heme transfer across the cell wall, we show that IsdC weakly binds heme (K D ‫؍‬ 0.34 ؎ 0.12 M) and that ZnPPIX rapidly dissociates from the protein at a rate of 126 ؎ 30 s ؊1. NMR studies of the apo-form of IsdC reveal that a 3 10 helix within the binding pocket undergoes a flexible to rigid transition as heme is captured. This structural plasticity may increase the efficiency of heme transfer across the cell wall by facilitating protein-protein interactions between apoIsdC and upstream hemoproteins.Staphylococcus aureus is an opportunistic Gram-positive pathogen that causes lethal infections such as toxic shock syndrome, meningitis, and endocarditis (1, 2). The bacterium needs the essential nutrient iron to grow and although the human body contains large quantities of this metal, little is directly available to S. aureus as it is sequestered intracellularly (3) or bound to transferrin and lactoferrin (4, 5). During infections, S. aureus procures iron from heme (protoporphyrin IX ϩ iron), which contains ϳ80% of the total iron in the body (6). Heme-loaded hemoglobin (Hb) 5 is released into the blood plasma by the action of microbial hemolysins that rupture erythrocytes (7). A group of newly discovered proteins called iron-regulated surface determinant (Isd) proteins then scavenge heme and transfer it into the cytoplasm where it is degraded to liberate iron (8, 9). The heme-binding IsdC protein plays an essential role in the transfer of heme across the cell wall peptidylglycan (6, 10). Proteins homologous to IsdC are also present in a number of other important human pathogens (Bacillus anthracis and Listeria monocytogenes) (6, 11). Therefore, compounds that inhibit their ability to capture heme may be useful antibiotics.In Gram-negative bacteria, the process of heme-iron acquisition is reasonably well understood. Heme is captured from hemoproteins or hemophore-hemoprotein complexes by specific outer membrane receptors (5). It is then transferred into the periplasm in a Ton-B-dependent manner, where it is moved across the inner membrane by specific ABC-dependent permeases (5). Heme acquisition mechanisms used by Gram-positive bacteria are only beginning...