Research
Crystallographic and Cryo-EM Studies of CHL1, SMN and Thioesterase I
I. CHL1
Nitrate transporter CHL1 from Arabidopsis is unique in that it was found to possess both high and low affinities to nitrate. CHL1 is a membrane protein which utilizes free energy stored in proton gradient to drive the transport of nitrate into cells. Sequence comparison has shown that this protein is similar to a peptide transporter family. However, CHL1 does not transport the peptides tested. In this project, we attempt to over-express and crystallize the CHL1 protein. Our investigation will assist in understanding the molecular mechanism of transporters in this family.
II. SMN
Spinal muscular atrophy (SMA) is a leading fatal autosomal recessive disease. Positional cloning has identified that survival motor neuron gene (SMN) is responsible for this disease. The SMN showed no sequence identity with previously identified genes. It is rather surprising to find that this protein is located in a subnuclear structure called gem (gemini of coiled bodies), implicating its function is related to RNA metabolism. Further investigation also shows this protein can associate with hnRNP or spliceosomal snRNAs, which are involved in RNA processing. The interaction of SMN with antiapoptotic factor Bcl 2 also suggests SMN may be involved in programmed cell death. We want to crystallize this protein and determine its structure. Information gained in this study will help us to understand how the SMN protein forms oligomers and how it interacts with other proteins and RNA. The structural information may also assist in finding a way to cure spinal muscular atrophy.
III. Thioesterase I
The Escherichia coli thioesterase I belongs to a discrete group of widely distributed esterases (ester hydrolases), which hydrolyze the esterase bond. It specifically catalyzes the hydrolytic cleavage of fatty acylcoenzyme A (CoA) thioesters and especially for the deacylation of the long acyl groups. It also can hydrolyze several short acyl-chain aromatic esters and short acyl-chain triacylglycerol. Interestingly thioesterase I has stereospecific synthesis and hydrolysis activities, which is expected to have industrial potential.
The crystal structure has been solved and refined to 1.9A resolution. We identified E. coli thioesterase I as one of a novel SGNH-hydrolase family. The unique GD-S-L-S motif is located at an (-loop and the first Ser is a nucleophilic residue. Based on the results of the mutational studies and the complex structure of diethyl pnitrophenol phosphate (DNP), the catalytic triad, Ser10-His157-Asp154, and oxyanion acceptors, Ser10-Gly44-Asn73, have been determined. Detailed structural studies of substrate binding pocket for stereospecific catalysis are undergoing.
