13 research outputs found
Structural insights into the function of the catalytically active human Taspase1
19 pags., 7 figs., 2 tabs.Taspase1 is an Ntn-hydrolase overexpressed in primary human cancers, coordinating cancer cell proliferation, invasion, and metastasis. Loss of Taspase1 activity disrupts proliferation of human cancer cells in vitro and in mouse models of glioblastoma. Taspase1 is synthesized as an inactive proenzyme, becoming active upon intramolecular cleavage. The activation process changes the conformation of a long fragment at the C-terminus of the α subunit, for which no full-length structural information exists and whose function is poorly understood. We present a cloning strategy to generate a circularly permuted form of Taspase1 to determine the crystallographic structure of active Taspase1. We discovered that this region forms a long helix and is indispensable for the catalytic activity of Taspase1. Our study highlights the importance of this element for the enzymatic activity of Ntn-hydrolases, suggesting that it could be a potential target for the design of inhibitors with potential to be developed into anticancer therapeutics.This project has been funded in whole with Federal funds from the National Cancer Institute (NCI), National Institutes of Health (NIH), under Chemical Biology Consortium contract no. HHSN261200800001E
Crystallographic studies of ligand binding by Zn-α_2-glycoprotein
Zn-α_2-glycoprotein (ZAG) is a 41 kDa soluble protein that is present in most bodily fluids. The previously reported 2.8 Å crystal structure of ZAG isolated from human serum demonstrated the structural similarity between ZAG and class I major histocompatibility complex (MHC) molecules and revealed a non-peptidic ligand in the ZAG counterpart of the MHC peptide-binding groove. Here we present crystallographic studies to explore further the nature of the non-peptidic ligand in the ZAG groove. Comparison of the structures of several forms of recombinant ZAG, including a 1.95 Å structure derived from ZAG expressed in insect cells, suggests that the non-peptidic ligand in the current structures and in the structure of serum ZAG is a polyethylene glycol (PEG), which is present in the crystallization conditions used. Further support for PEG binding in the ZAG groove is provided by the finding that PEG displaces a fluorophore-tagged fatty acid from the ZAG binding site. From these results we hypothesize that our purified forms of ZAG do not contain a bound endogenous ligand, but that the ZAG groove is capable of binding hydrophobic molecules, which may relate to its function
The Mobility of a Conserved Tyrosine Residue Controls Isoform-Dependent Enzyme–Inhibitor Interactions in Nitric Oxide Synthases
Many pyrrolidine-based inhibitors
highly selective for neuronal
nitric oxide synthase (nNOS) over endothelial NOS (eNOS) exhibit dramatically
different binding modes. In some cases, the inhibitor binds in a 180°
flipped orientation in nNOS relative to eNOS. From the several crystal
structures we have determined, we know that isoform selectivity correlates
with the rotamer position of a conserved tyrosine residue that H-bonds
with a heme propionate. In nNOS, this Tyr more readily adopts the
out-rotamer conformation, while in eNOS, the Tyr tends to remain fixed
in the original in-rotamer conformation. In the out-rotamer conformation,
inhibitors are able to form better H-bonds with the protein and heme,
thus increasing inhibitor potency. A segment of polypeptide that runs
along the surface near the conserved Tyr has long been thought to
be the reason for the difference in Tyr mobility. Although this segment
is usually disordered in both eNOS and nNOS, sequence comparisons
and modeling from a few structures show that this segment is structured
quite differently in eNOS and nNOS. In this study, we have probed
the importance of this surface segment near the Tyr by making a few
mutants in the region followed by crystal structure determinations.
In addition, because the segment near the conserved Tyr is highly
ordered in iNOS, we also determined the structure of an iNOS–inhibitor
complex. This new structure provides further insight into the critical
role that mobility plays in isoform selectivity