(Acyloxy)alkoxy moiety as amino acids protecting group for the synthesis of (R,R)-2,7 diaminosuberic acid via RCM.

A new synthetic pathway is described to prepare asymmetrically protected 2,7-diaminosuberic acid. This strategy exploits (acyloxy)alkoxy promoiety as protecting group and RCM reaction using second generation Grubbs catalyst and provides the trans isomer of (2R,7R)-7-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-(tert-butoxycarbonylamino)-8- methoxy-8-oxooct-4-enoic acid, which was in turn reduced to obtain (2R,7R)-7-(((9H-fluoren-9-yl)methoxy)carbonylamino)- 2-(tert-butoxycarbonylamino)-8-methoxy-8-oxooctanoic acid.

The structure of the Staphylococcus aureus sortase-substrate complex reveals how the universally conserved LPXTG sorting signal is recognized.

In Gram-positive bacteria, sortase enzymes assemble surface proteins and pili in the cell wall envelope. Sortases catalyze a transpeptidation reaction that joins a highly conserved LPXTG sorting signal within their polypeptide substrate to the cell wall or to other pilin subunits. The molecular basis of transpeptidation and sorting signal recognition are not well understood, because the intermediates of catalysis are short lived. We have overcome this problem by synthesizing an analog of the LPXTG signal whose stable covalent complex with the enzyme mimics a key thioacyl catalytic intermediate. Here we report the solution structure and dynamics of its covalent complex with the Staphylococcus aureus SrtA sortase. In marked contrast to a previously reported crystal structure, we show that SrtA adaptively recognizes the LPXTG sorting signal by closing and immobilizing an active site loop. We have also used chemical shift mapping experiments to localize the binding site for the triglycine portion of lipid II, the second substrate to which surface proteins are attached. We propose a unified model of the transpeptidation reaction that explains the functions of key active site residues. Since the sortase-catalyzed anchoring reaction is required for the virulence of a number of bacterial pathogens, the results presented here may facilitate the development of new anti-infective agents.

NMR structure of the amino-terminal domain of the lambda integrase protein in complex with DNA: immobilization of a flexible tail facilitates beta-sheet recognition of the major groove.

The integrase protein (Int) from bacteriophage lambda is the archetypal member of the tyrosine recombinase family, a large group of enzymes that rearrange DNA in all domains of life. Int catalyzes the insertion and excision of the viral genome into and out of the Escherichia coli chromosome. Recombination transpires within higher-order nucleoprotein complexes that form when its amino-terminal domain binds to arm-type DNA sequences that are located distal to the site of strand exchange. Arm-site binding by Int is essential for catalysis, as it promotes Int-mediated bridge structures that stabilize the recombination machinery. We have elucidated how Int is able to sequence specifically recognize the arm-type site sequence by determining the solution structure of its amino-terminal domain (Int(N), residues Met1 to Leu64) in complex with its P'2 DNA binding site. Previous studies have shown that Int(N) adopts a rare monomeric DNA binding fold that consists of a three-stranded antiparallel beta-sheet that is packed against a carboxy-terminal alpha helix. A low-resolution crystal structure of the full-length protein also revealed that the sheet is inserted into the major groove of the arm-type site. The solution structure presented here reveals how Int(N) specifically recognizes the arm-type site sequence. A novel feature of the new solution structure is the use of an 11-residue tail that is located at the amino terminus. DNA binding induces the folding of a 3(10) helix in the tail that projects the amino terminus of the protein deep into the minor groove for stabilizing DNA contacts. This finding reveals the structural basis for the observation that the "unstructured" amino terminus is required for recombination.

The IsdC protein from Staphylococcus aureus uses a flexible binding pocket to capture heme.

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 angstroms. IsdC partially buries protoporphyrin within a large hydrophobic pocket that is located at the end of its beta-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 microm) 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.

Solution structure of the NEAT (NEAr Transporter) domain from IsdH/HarA: the human hemoglobin receptor in Staphylococcus aureus.

During infections the pathogen Staphylococcus aureus procures the essential nutrient iron from its host using iron-regulated surface determinant (Isd) proteins, which scavenge heme bound iron from host hemoproteins. Four Isd proteins are displayed in the cell wall, where they function as receptors for host proteins and heme. Each of the receptors contains one or more copies of a recently discovered domain called NEAT (NEAr Transporter) that has been shown to mediate protein binding. Here we report the three-dimensional solution structure of the NEAT domain from the IsdH/HarA protein, which is the hemoglobin receptor in the Isd system. This is the first structure of a NEAT domain and reveals that they adopt a beta sandwich fold that consists of two five-stranded antiparallel beta sheets. Although unrelated at the primary sequence level, our results indicate that NEAT domains belong to the immunoglobulin superfamily. Binding studies indicate that two IsdH/HarA NEAT domains bind a single molecule of methemoglobin, while the distantly related NEAT domain from the S. aureus IsdC protein binds only heme. A comparison of their primary sequences in light of the new structure is used to predict the hemoglobin and heme binding surfaces on NEAT domains.

Synthesis and structural modeling of the amphiphilic siderophore rhizobactin-1021 and its analogs.

We describe two convenient syntheses of rhizobactin-1021 (Rz), a citrate-based siderophore amphiphile produced by the nitrogen-fixing root symbiont Rhizobium meliloti-1021, and several analogs. Our approach features a singly amidated, tert-butyl-protected citrate intermediate that easily affords a variety of Rz analogs in the late stages of the synthesis. Structural modeling and the monolayer behavior of Rz and its metal complexes are consistent with a structural reorganization upon Rz-mediated iron chelation.

Membrane dynamics of the amphiphilic siderophore, acinetoferrin.

Acinetobacter haemolyticus is an antibiotic resistant, pathogenic bacterium responsible for an increasing number of hospital infections. Acinetoferrin (Af), the amphiphilic siderophore isolated from this organism, contains two unusual trans-2-octenoyl hydrocarbon chains reminiscent of a phospholipid structural motif. Here, we have investigated the membrane affinity of Af and its iron complex, Fe-Af, using small and large unilamellar phospholipid vesicles (SUV and LUV) as model membranes. Af shows a high membrane affinity with a partition coefficient, K(x)= 6.8 x 10(5). Membrane partitioning and trans-membrane flip-flop of Fe-Af have also been studied via fluorescence quenching of specifically labeled vesicle leaflets and (1)H NMR line-broadening techniques. Fe-Af is found to rapidly redistribute between lipid and aqueous phases with dissociation/partitioning rates of k(off) = 29 s(-1) and k(on) = 2.4 x 10(4) M(-1) s(-1), respectively. Upon binding iron, the membrane affinity of Af is reduced 30-fold to K'(x) = 2.2 x 10(4) for Fe-Af. In addition, trans-membrane flip-flop of Fe-Af occurs with a rate constant, k(p) = 1.2 x 10(-3) s(-1), with egg-PC LUV and a half-life time around 10 min with DMPC SUV. These properties are due to the phospholipid-like conformation of Af and the more extended conformation of Fe-Af that is enforced by iron binding. Remarkable similarities and differences between Af and another amphiphilic siderophore, marinobactin E, are discussed. The potential biological implications of Af and Fe-Af are also addressed. Our approaches using inner- and outer-leaflet-labeled fluorescent vesicles and (1)H NMR line-broadening techniques to discern Af-mediated membrane partitioning and trans-membrane diffusion are amenable to similar studies for other paramagnetic amphiphiles.

Mycobactin-mediated iron acquisition within macrophages.

Restricting the availability of iron is an important strategy for defense against bacterial infection. Mycobacterium tuberculosis survives within the phagosomes of macrophages; consequently, iron acquisition is particularly difficult for M. tuberculosis, because the phagosomal membrane is an additional barrier for its iron access. However, little is known about the iron transport and acquisition pathways adapted by this microbe in vivo. Extracellular iron sources are usually mobilized by hydrophilic siderophores. Here, we describe direct evidence that mycobactins, the lipophilic siderophores of mycobacteria, efficiently extract intracellular macrophage iron. The metal-free siderophore is diffusely associated with the macrophage membrane, ready for iron chelation. Notably, the mycobactin-metal complex accumulates with high selectivity in macrophage lipid droplets, intracellular domains for lipid storage and sorting. In our experiments, these mycobactin-targeted lipid droplets were found in direct contact with phagosomes, poised for iron delivery. The existence of this previously undescribed iron acquisition pathway indicates that mycobacteria have taken advantage of endogenous macrophage mechanisms for iron mobilization and lipid sorting for iron acquisition during infection. The pathway could represent a new target for the control of mycobacterial infection.

Synthesis, structure, and molecular dynamics of gallium complexes of schizokinen and the amphiphilic siderophore acinetoferrin.

A new general synthesis of the citrate-based siderophores acinetoferrin (Af) and schizokinen (Sz) and their analogues is described. The molecular structure of gallium schizokinen, GaSz, was determined by combined (1)H NMR, Hartree-Fock ab initio calculations, DFT, and empirical modeling of vicinal proton NMR spin-spin couplings. The metal-coordination geometry of GaSz was determined from NOE contacts to be cis-cis with respect to the two chelating hydroxamates. One diaminopropane adopts a single chairlike conformation while another is a mixture of two ring pucker arrangements. Both amide hydrogens are internally hydrogen bonded to metal-ligating oxygen atoms. The acyl methyl groups are directed away from each other with an average planar angle of ca. 130 degrees. The kinetics of GaSz racemization were followed by selective, double spin-echo inversion-recovery (1)H NMR spectroscopy over the temperature range of 10-45 degrees C. The racemization proceeds by a multistep mechanism that is proton independent between pD 5 and 12 (k(0) = 1.47 (0.15 s(-1))) and acid catalyzed below pD 4 (k(1) = 2.25 (0.15) x 10(4) M(-1) s(-1)). The activation parameters found for the two sequential steps of the proton independent pathway were DeltaH(++) = 25 +/- 3 kcal M(-1), DeltaS(++) = 25 +/- 7 cal M(-1) K(-1) and DeltaH(++) = 17.1 +/- 0.2 kcal M(-1), DeltaS(++) = 0.3 +/- 2.7 cal M(-1) K(-1). The first step of the proton-independent mechanism was assigned to the dissociation of the carboxyl group. The second step was assigned to complex racemization. The proton-assisted step was assigned to a complete dissociation of the alpha-hydroxy carboxyl group at pD < 4. The ab initio modeling of gallium acinetoferrin, GaAf, and analogues derived from the structure of GaSz has shown that the pendant trans-octenoyl fragments are oriented in opposite directions with the average planar angle of ca. 130 degrees. This arrangement prevents GaAf from adopting a phospholipid-like structural motif. Significantly, iron siderophore complex FeAf was found to be disruptive to phospholipid vesicles and is considerably more hydrophilic than Af, with an eight-fold smaller partition coefficient.