3EGY

Crystal Structure of Human Thymidyalte Synthase A191K with Loop 181-197 stabilized in the inactive conformation

Summary for 3EGY

• Entry DOI: 10.2210/pdb3egy/pdb
• Related: 1HVY 1HW3 1YPV 2ONB 2RD8 2RDA 3EHI
• Descriptor: Thymidylate synthase, SULFATE ION, 1,2-ETHANEDIOL, ... (4 entities in total)
• Functional Keywords: methyltransferase, nucleotide biosynthesis, transferase
• Biological source: Homo sapiens (human)
• Cellular location: Nucleus : P04818
• Total number of polymer chains: 1
• Total formula weight: 36379.70

Authors

Lovelace, L.L.,Gibson, L.M.,Johnson, S.R.,Berger, S.H.,Lebioda, L. (deposition date: 2008-09-11, release date: 2009-08-18, Last modification date: 2023-08-30)

Primary citation

Lovelace, L.L.,Johnson, S.R.,Gibson, L.M.,Bell, B.J.,Berger, S.H.,Lebioda, L.
Variants of human thymidylate synthase with loop 181-197 stabilized in the inactive conformation.
Protein Sci., 18:1628-1636, 2009

PubMed Abstract: Loop 181-197 of human thymidylate synthase (hTS) populates two major conformations, essentially corresponding to the loop flipped by 180 degrees . In one of the conformations, the catalytic Cys195 residue lies distant from the active site making the enzyme inactive. Ligands stabilizing this inactive conformation may function as allosteric inhibitors. To facilitate the search for such inhibitors, we have expressed and characterized several mutants designed to shift the equilibrium toward the inactive conformer. In most cases, the catalytic efficiency of the mutants was only somewhat impaired with values of k(cat)/K(m) reduced by factors in a 2-12 range. One of the mutants, M190K, is however unique in having the value of k(cat)/K(m) smaller by a factor of approximately 7500 than the wild type. The crystal structure of this mutant is similar to that of the wt hTS with loop 181-197 in the inactive conformation. However, the direct vicinity of the mutation, residues 188-194 of this loop, assumes a different conformation with the positions of C(alpha) shifted up to 7.2 A. This affects region 116-128, which became ordered in M190K while it is disordered in wt. The conformation of 116-128 is however different than that observed in hTS in the active conformation. The side chain of Lys190 does not form contacts and is in solvent region. The very low activity of M190K as compared to another mutant with a charged residue in this position, M190E, suggests that the protein is trapped in an inactive state that does not equilibrate easily with the active conformer.

PubMed: 19569192
DOI: 10.1002/pro.171

Experimental method

X-RAY DIFFRACTION (2.18 Å)