Dr. Ramin Radfar

Structural Chemistry and Protein Crystallography

Knowledge of atomic resolution structure provides tremendous insight into understanding of how the protein functions and structure-function relationships. Our research is focused on determination of 3-dimensial molecular structure of biologically important proteins and/or enzyme-inhibitor complexes using x-ray diffraction.

Folate and its derivatives are involved in several metabolic areas, both biosynthetic and degradative pathways. Many of the reactions are common to most metabolically active cells, but there are various folate-dependent reactions that are species and/or tissue specific. Tetrahydrofolate (THF) and its derivatives are the biologically active forms of folic acid, a four-electron oxidized form of THF. They are specialized co-substrates for a variety of enzymes involved in one-carbon metabolism. THF reacts in an ATP-dependent manner with formate in a reaction catalyzed by formyltetrahydrofolate synthetase (FTHFS). 

Crystal structure of FTHFS was solved by using multiwavelength anomalous dispersion method. The FTHFS protomer containing 559 residues consists of one large and two smaller domains. Domain one includes residues 7-120 and 254-431. The core of this domain is formed from a central sheet with seven parallel strands (b4, b5, b14-17). Strand b13 is almost perpendicular to the plane of the core sheet and links domains two to one. The core sheet is surrounded by eight a-helices (a1,a3,a10-15), three b-strands (b1-3), and the a/b motif (a4-5, b6-7) that connects domains one to two. Domain two includes residues 131-250 and is composed of five b- strands (b8-12) and four a-helices (a6-9). Domain three with 124 residues (433-557) contains six b-strands (b19-24) and two a-helices (a16-17).

The deep cleft between the domains is likely the active site pocket and the binding site for ATP, formate, and THF. The N- (residue 7) and C-termini (residue 557) are just 16 apart; the subunit possesses approximate dimensions of 70 50 40 . Studies of FTHFS catalysis showed a strong enhancement of catalytic activity by ammonium ions, manifested as a decrease in the Km of formate with increasing ammonium ion concentration. At saturating levels of ammonium ion, a 10-fold decrease in the Km for formate has been reported. Enzymes isolated from both purinolytic organisms and acetogens show this effect. The efficacy of the particular cation in question seems to be, at least in part, a function of its individual ionic radius. The impact of monovalent cations on catalysis by FTHFS is greatest for the ammonium ion and decreases in the order: NH4+ > K+ = Rb+ > Cs+ > Na+ = Li+.

Structure of FTHFS-Cs+/K+ complexes were determined by using the original structure of FTHFS, determined in the presence of ammonium cation, which has been refined to 2.5 resolution. For the cesium ion complex the resulting electron density map showed two strong peaks (14.6 and 12.1 sigma for subunit A and B, respectively), which we assigned to cesium ions. The cesium cation is in contact with the OE1 oxygen of glutamate 98 and the carbonyl oxygen atoms of Pro 99, Ile 125, Asn 126, Leu 127 and two water molecules. The protein residues forming the cesium ion-binding site belong to two coils; the first coil connects helices 5 and 6 in domain one while the other coil links b-strand 5 and a-helix 4 in domain two. This part of the molecule is approximately at the interface between the two more tightly bound monomers. The cation ligands, however, are all from the same subunit; thus the cation is not involved in crosslinking to the other subunit.

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Prepared and Last Updated 09.2015 by Ramin Radfar

Contact information: Milliken Science Center 309, 429 N. Church Street, Spartanburg, SC 29303. Tel: (864) 597-4632