Human neuron specific enolase, chicken/human annexin V, formyltetrahydrofolate synthetase: Structural and mechanistic study

Human neuron specific enolase (hNSE) is a major protein of the brain, constituting 2--4% of soluble protein in neurons. Here, we present the expression, purification, crystallographic structures and kinetic studies of hNSE. We have determined hNSE native structure, and structure of the hNSE complex with natural substrate/product (PGA/PEP). Also studies of several inhibitory complexes are reported. The structures of complex between hNSE and two pairs of inhibitors TSP/AEP, TSP/LAP are the first documented cases in which a homodimer binds two different inhibitors forming asymmetric structure. The study of human neuron specific enolase clearly shows that during catalysis, the two subunits of enolase become asymmetric and work together in a dynamical/interactive way to facilitate the substrate dehydration/hydration and product release. The concepts from this study can be used to design more efficient heteroinhibitory systems that exploit the cooperativity of asymmetric enzymes.;Annexin V is a member of annexins family that binds to phospholipid membranes in a calcium-dependent manner. The in vivo functions of annexin V include roles in exocytosis and membrane trafficking, Ca2+-channel activity, membrane fusion, etc. We have studied how chicken annexin V crystal structures are affected by Ca2+, Zn2+, Ca 2+/Zn2+, GTP, ATP, L-alpha-lysophosphatidyl-serine and low pH. Crystal structures of human annexin V with O-phospho-serine and phosphatidyl inositol were also investigated. We observed that Zn2+ helps to organize annexin V molecules in a different arrangement which disrupts the requirements for Ca2+-dependent binding to membrane phospholipids. We also determined the O-phospho-serine binding site in human annexin V and L-alpha-lysophosphatidyl-serine binding site in chicken annexin V. Both structures indicate that Ca2+ acts as the bridge to link the annexin V with the phopholipid headgroup in the membrane.;Formyltetrahydrofolate synthetase (FTHFS) is an important folate-utilizing enzyme. Its function in most tissues is to incorporate formate into the active one-carbon pool. We have successfully crystallized FTHFS from Moorella thermoacetica (FTHFS-m.t.) and from Clostridium cylindrosporum (FTHFS-c.c.) both in high salt and low salt conditions. The structures of FTHFS complexes with ATP or ZD9331 have been solved to 2.50 A, 2.56 A resolution, respectively. We observed the asymmetrically bound ligand molecule in both FTHFS complexes.