Packaging of cyclophilin A (CypA) into HIV-1 virions is essential for efficient replication; however, the reason for this is unknown. Incorporation is mediated through binding to the Gly-89 -Pro-90 peptide bond of the N-terminal domain of HIV-1 capsid (CA N ). Despite the fact that CypA is a peptidyl-prolyl cis͞trans isomerase, catalytic activity on CA N has not been observed previously. We show here, using NMR exchange spectroscopy, that CypA does not only bind to CA N but also catalyzes efficiently the cis͞trans isomerization of the Gly-89 -Pro-90 peptide bond. In addition, conformational changes in CA N distal to the CypA binding loop are observed on CypA binding and catalysis. The results provide experimental evidence for efficient CypA catalysis on a natively folded and biologically relevant protein substrate.

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The camphor hydroxylase cytochrome P450(cam) (CYP101) catalyzes the 5-exo hydroxylation of camphor in the first step of camphor catabolism by Pseudomonas putida. CYP101 forms a specific electron transfer complex with its physiological reductant, the Cys(4)Fe(2)S(2) ferredoxin putidaredoxin (Pdx). Pdx, along with other proteins and small molecules, has also been shown to be an effector for turnover by CYP101. Multidimensional nuclear magnetic resonance (NMR) techniques have been used to make extensive sequential (1)H, (15)N, and (13)C resonance assignments in CYP101 that permit a more complete characterization of the complex formed by CYP101 and Pdx. NMR-detected perturbations in CYP101 upon Pdx binding encompass regions of the CYP101 remote from the putative Pdx binding site, including in particular a region of the CYP101 molecule that has been implicated in substrate access to the active site via dynamical processes. A model for effector activity is proposed in which the primary role of the effector is to prevent uncoupling (formation of reduced oxo species without formation of hydroxycamphor) by enforcing conformations of CYP101 that prevent loss of substrate and/or intermediates prior to turnover. A secondary role could also be to enforce conformations that permit efficient proton transfer into the active site for coupled proton/electron transfer.

Helicobacter pylori, a pathogen that colonizes the human stomach, requires the nickel-containing metalloenzymes urease and NiFe-hydrogenase to survive this low pH environment. The maturation of both enzymes depends on the metallochaperone, HypA. HypA contains two metal sites, an intrinsic zinc site and a low-affinity nickel binding site. X-ray absorption spectroscopy (XAS) shows that the structure of the intrinsic zinc site of HypA is dynamic, and able to sense both nickel loading and pH changes. At pH 6.3, an internal pH that occurs during acid shock, the zinc site undergoes unprecedented ligand substitutions to convert from a Zn(Cys) 4 site to a Zn(His) 2 (Cys) 2 site. NMR spectroscopy shows that binding of Ni(II) to HypA results in paramagnetic broadening of resonances near the N-terminus. NOEs between the β-CH 2 protons of Zn cysteinyl ligands are consistent with a strand-swapped HypA dimer. Addition of nickel causes resonances from zinc binding motif and other regions to double, indicating more than one conformation can exist in solution. Although the structure of the high-spin, 5-6 coordinate Ni(II) site is relatively unaffected by pH, the nickel binding stoichiometry is decreased from one per monomer to one per dimer at pH = 6.3. Mutation of any cysteine residue in the zinc binding motif results in a zinc site structure similar to that found for holo-WT-HypA at low pH and is unperturbed by the addition of nickel. Mutation of the histidines that flank the CXXC motifs results in a zinc site structure that is similar to holo-WT-HypA at neutral pH (Zn(Cys) 4 ) and is no longer responsive to nickel binding or pH changes. Using an in vitro urease activity assay, it is shown that the recombinant protein is sufficient for recovery of urease activity in cell lysate from a HypA deletion mutant, and that mutations in the zinc-binding motif result in a decrease in recovered urease activity. The results are interpreted in terms of a model wherein HypA controls the flow of nickel traffic in the cell in response to nickel availability and pH. KeywordsHelicobacter pylori; XAS; HypA; metallochaperone; zinc; nickel; ITC; NMR mmaroney@chemistry.umass.edu. Supporting Information Available: Figures of CD spectra for zinc-site cysteine mutants of HypA, Thermal melts of WT-and zinc-site cysteine and histidine mutants, molecular weight determinations by size-exclusion chromatography, ITC thermograms for zinc-site cysteine and histidine mutants, raw ITC titration data, zinc K-edge XANES and EXAFS data and fits for Cys → Asp and His95A mutations, nickel K-edge XANES and EXAFS data and fits for Cys → Asp zinc-site mutations, and UV-vis spectra of HypA with nickel bound. Tables of mutagenic primers, best EXAFS fits to Zn K-edge data for Cys → Asp mutations, best EXAFS fits to Ni Kedge data for zinc-site Cys → Asp mutations, alternate fits for zinc and nickel K-edge EXAFS (39 pages). This information is available free of charge via the Internet at

SummaryAcireductone dioxygenase (ARD) catalyzes different reactions between O 2 and 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene (acireductone) depending upon the metal bound in the active site. Ni +2 -ARD cleaves acireductone to formate, CO and methylthiopropionate. If Fe +2 is bound (ARD ′), the same substrates yield methylthioketobutyrate and formate. The two forms differ in structure, and are chromatographically separable. Paramagnetism of Fe +2 renders the active site of ARD′ inaccessible to standard NMR methods. The structure of ARD′ has been determined using Fe +2 binding parameters determined by X-ray absorption spectroscopy and NMR restraints from H98S ARD, a metal-free diamagnetic protein that is isostructural with ARD′. ARD′ retains the β-sandwich fold of ARD, but a structural entropy switch increases order at one end of a two-helix system that bisects the β-sandwich and decreases order at the other upon interconversion of ARD and ARD′, causing loss of the C-terminal helix in ARD′ and rearrangements of residues involved in substrate orientation in the active site.

Here we report the structure of acireductone dioxygenase (ARD), the first determined for a new family of metalloenzymes. ARD represents a branch point in the methionine salvage pathway leading from methylthioadenosine to methionine and has been shown to catalyze different reactions depending on the type of metal ion bound in the active site. The solution structure of nickel-containing ARD (Ni-ARD) was determined using NMR methods. X-ray absorption spectroscopy, assignment of hyperfine shifted NMR resonances and conserved domain homology were used to model the metal-binding site because of the paramagnetism of the bound Ni2+. Although there is no structure in the Protein Data Bank within 3 A r.m.s deviation of that of Ni-ARD, the enzyme active site is located in a conserved double-stranded b-helix domain. Furthermore, the proposed Ni-ARD active site shows significant post-facto structural homology to the active sites of several metalloenzymes in the cupin superfamily.

The two-protein complex between putidaredoxin (Pdx) and cytochrome P450(cam) (CYP101) is the catalytically competent species for camphor hydroxylation by CYP101. We detected a conformational change in CYP101 upon binding of Pdx that reorients bound camphor appropriately for hydroxylation. Experimental evidence shows that binding of Pdx converts a single X-proline amide bond in CYP101 from trans or distorted trans to cis. Mutation of proline 89 to isoleucine yields a mixture of both bound camphor orientations, that seen in Pdx-free and that seen in Pdx-bound CYP101. A mutation in CYP101 that destabilizes the cis conformer of the Ile 88-Pro 89 amide bond results in weaker binding of Pdx. This work provides direct experimental evidence for involvement of X-proline isomerization in enzyme function.

Removal of substrate (+)-camphor from the active site of cytochrome P450cam (CYP101A1) results in nuclear magnetic resonance-detected perturbations in multiple regions of the enzyme. The 1H,15N correlation map of substrate-free diamagnetic Fe(II) CO-bound CYP101A permits these perturbations to be mapped onto the solution structure of the enzyme. Residual dipolar couplings (RDCs) were measured for 15N-1H amide pairs in two independent alignment media for the substrate-free enzyme and used as restraints in solvated molecular dynamics (MD) simulations to generate an ensemble of best-fit structures of the substrate-free enzyme in solution. NMR-detected chemical shift perturbations reflect changes in the electronic environment of the NH pairs, such as hydrogen bonding and ring current shifts, and are observed for residues in the active site as well as in hinge regions between secondary structural features. RDCs provide information regarding relative orientations of secondary structures, and RDC-restrained MD simulations indicate that portions of a β-rich region adjacent to the active site shift so as to partially occupy the vacancy left by removal of substrate. The accessible volume of the active site is reduced in the substrate-free enzyme relative to the substrate-bound structure calculated using the same methods. Both symmetric and asymmetric broadening of multiple resonances observed upon substrate removal as well as localized increased errors in RDC fits suggest that an ensemble of enzyme conformations are present in the substrate-free form.