Research interests

Our research spans the fields of biochemistry and biophysics. We are interested in the role of metal ions in protein folding and molecular recognition. To understand these basic phenomena we use a variety of techniques including UV/vis, fluorescence, and CD spectroscopy, microscopy, X-ray based techniques, and NMR methods. Currently, work in our lab is divided between two areas of interest: host cell:HIV interactions and peroxisomal protein targeting.

Biochemical Studies of HIV-1 Virion Infectivity Factor

Virion infectivity factor (Vif) is a 23 kD accessory protein that promotes the ubiquitination and proteasomal degradation of Apo3G, a cellular cytidine deaminase. Interaction of Vif with a Cullin 5 (Cul5)-based E3 ligase is required for Apo3G degradation and recent studies suggest that a conserved HCCH motif plays a vital role in the Vif-Cul5 interaction (Xiao et al.; Mehle et al., 2006). The only other documented case of Cul5 hijacking involves the human papilloma virus oncoprotein E6 (E4orf6). E4orf6 acts in concert with the protein E6-AP to target and degrade p53. E4orf6 contains two copies of the unusual Cys-rich motifs (C-X2-C-X29-C-X2-C) in its N and C termini. The recent solution structure of the C-terminal domain of E4orf6 (2006 Mol Cell 21:665) reveals that this motif binds zinc to stabilize a unique α/β fold that exposes several clusters of conserved residues to the solvent. We are using several biochemical and biophysical approaches to unravel the mechanism of Vif. Cullins act as molecular scaffolds to assemble a functionally diverse array of CRL complexes. Viral "hijacking" of CRLs represents a common mechanism of immune evasion by which specific cellular proteins are targeted for destruction. Although there are many examples of viral proteins that redirect CRL activity, very few involve the Cul5-based CRL.Download a pdf summary


Peroxisomal Protein Trafficking

Peroxisomes are found in eukaryotes. Peroxisomal targeting signal-1 (PTS1) is a conserved tripeptide that is found at the C-terminus of more than 95% of all known peroxisomal matrix proteins. Pex5 is the PTS1 receptor and contains 7 conserved tetratricopeptide repeat (TPR) domains at its C terminus. We are studying how PTS1 binding to Pex5 is signaled to other downstream components (Pex14, Pex10, and Pex12) of the import machinery. Recent structural work suggests that a key to the Pex5 mechanism involves intrinsic disorder in Pex5. We are studying molecular dynamics in the N- and C-termini of Pex5 using fluorescence techniques.Download a pdf summary


Recent work published

"Molecular Structure and Biochemical Properties of the HCCH-Zn2+ Site in HIV-1 Vif" » In this study, the zinc binding properties and molecular structure of the zinc binding site in HIV-1 Vif were examined using various methods including X-ray absorption spectroscopy (XAS). XAS revealed that the HCCH motif in wild-type Vif (modeled using the peptide HCCHp) coordinates zinc using the ligands His108, Cys114, Cys133, and His139. This was not unexpected but our study is the first to directly analyze zinc coordination by Vif. It was surprising that mutation of one or more of the zinc ligands did not significantly affect the zinc binding affinity of HCCHp (Kd values for the HCCHp-Zn2+ complex ranged from 50 nM to 800 nM). XAS revealed that the mutants, C114S, C133S, and C114/133S, coordinate zinc using alternate N/O ligands. These mutations abolish Vif function presumably by altering the conformation of Vif and preventing association with the E3 ligase that induces the polyubiquitination and subsequent proteosomal degradation of APOBEC3G. Importantly, we demonstrated a direct interaction between HCCHp and human Cul5 and showed that this interaction is enhanced nearly 10-fold in the presence of zinc. Ongoing work is focused on the Vif-Cul5 interaction.
Biochemistry (2009) xx:xxx

"Conformational analysis of a peptide approximating the HCCH motif in HIV-1 Vif" » Virion infectivity factor (Vif) is an accessory protein encoded by HIV-1. Vif recruits a Cul5-based ubiquitin ligase that targets APOBEC3G, a host-encoded antiviral enzyme, for proteasomal degradation. The C-terminus of Vif contains a conserved His-X(5)-Cys-X(17-18)-Cys-X(3-5)-His (HCCH) motif that binds zinc and interacts with Cul5. In this study, CD spectroscopy, fluorescence spectroscopy, light scattering, and zinc binding assays were used to examine the conformational properties of HCCHp, a 42-amino acid peptide encompassing the HCCH motif. A single tryptophan residue was engineered into HCCHp to probe local structural changes induced by zinc binding. Zinc binding increased burial of the Trp residue from solvent and increased tertiary packing. The solvent 2,2,2-trifluoroethanol (TFE) induced the formation of an alpha-helical conformation of HCCHp with a midpoint of 20% (vol/vol) and inhibited zinc-induced aggregation of HCCHp. TFE titration data were sigmoidal, consistent with the cooperative nature of helix formation. Zinc binding to HCCHp in 30% TFE solutions was cooperative and weakened the TFE-induced structure. In 80% TFE solutions this cooperativity was lost, suggesting a mechanism in which monomeric and oligomeric peptide forms display different affinities for zinc. TFE weakened zinc binding to HCCHp by two orders of magnitude relative to the zinc binding affinity measured in aqueous solvent. The data suggest that HCCHp conformation and zinc binding affinity are tightly coupled. We propose that the lack of intrinsic structure in the HCCH motif may be important for Vif's function as an E3 ubiquitin ligase adaptor protein.
Biopolymers: Peptide Science (2009) 92:xxx

"Zinc binding regulates conformation of HIV-1 Vif"
Vif, a key regulator of HIV infectivity, contains a highly conserved HCCH motif. Recently we characterized the metal binding properties of this motif. Our primary discovery was that the HCCH motif binds zinc with high specificity. Zinc binding induces a protein conformational change that mediates protein-protein interactions. This conformational change is likely important for Vif function and is currently being studied.
Proc. Nat. Acad. Sci. (2006) 103:18475

"Affinity threshold determines efficiency of peroxisomal protein targeting"
In a recent report, we quantitated the binding of various PTS1 ligands to the human Pex5 protein (Pex5p) and two mutant variants of Pex5p, Pex5p(N489K) and Pex5p(S563W). The two variants are interesting in that they are responsible for two versions of peroxisomal disorders in humans with significantly different severities, relative to complete inactivation of Pex5p, namely infantile Refsum disease (IRD) and neonatal adreno-leukodystrophy (NALD). Complete malfunction of the Pex5 protein results in yet a third variant, the most severe disease, Zellweger syndrome. The results of our study suggest that the differences between these syndromes is due to differences in the ability of their Pex5s to bind to PTS1, with Zellweger syndrome the most severe with total loss of PTS1 and PTS2 binding ability, NALD the next most severe with elimination of PTS1 binding but continued PTS2 binding and IRD with only a partial loss of binding of certain PTS1 containing proteins. This study explains in molecular terms why mutations in one gene (PEX5) can lead to diseases with three very distinct phenotypes.
J. Mol. Biol.(2007) 368:1259