STRUCTURES OF THERMUS THERMOPHILUS ELONGATION FACTOR TU MUTANTS LACKING CONSERVED HISTIDINE RESIDUES
Tanis Hogg1,
Jeroen R. Mesters1, Annett Wagner1,2,
Mathias Sprinzl2 and Rolf Hilgenfeld1
1 Institute
of Molecular Biotechnology, Department of Structural Biology and
Crystallography, Beutenbergstrasse 11, D-07745 Jena, Germany,
e-mail: thogg@imb-jena.de
2 Laboratorium für
Biochemie, Universität Bayreuth, Universitätsstrasse 30,
D-95440 Bayreuth, Germany
Keywords: EF-Tu, protein biosynthesis, mutant structures
In order to accomplish its role
in protein biosynthesis, prokaryotic elongation factor Tu (EF-Tu)
shuttles through a complex cycle where it makes critical
interactions with several different molecules. In most cases,
these interactions are secured by means of specific residues
which are highly conserved throughout different species. Two
evolutionarily well-conserved histidine residues, His-67 and
His-85, are suspected to maintain certain binding and catalytic
properties of EF-Tu during this cycle. His-67 is fully conserved
in prokaryotic EF-Tu and archaebacterial EF-1, with the exception
of the prokaryotic chloroplast EF-Tu's (His->Thr) and the
selenocysteine-specific elongation factor SelB, which carries a
tyrosine residue at the equivalent position (1). In the GTP-bound
state, His-67 forms part of the aa-tRNA binding site, where it is
proposed to play a major role in stabilizing the ternary complex.
Completely conserved in prokaryotic EF-Tu's, His-85 is situated
in the "Switch II" region (residues 81-101). In the
GTP-bound conformation, His-85 resides adjacent to the nucleotide
binding pocket and is suggested to be involved in
ribosome-stimulated GTP-hydrolysis (2). Extensive biochemical
characterizations and mutational studies have been conducted in
order to understand the specific activity of these residues
(3-6), yet a direct mechanistic relationship between a single
mutation and the observed biochemical changes cannot always be
established (7). Accordingly, we have recently determined the
X-ray crystal structures of two such mutants, His67Ala and
His85Leu, analyzed to 2.7 A and 2.6 A resolution respectively.
Solved in their GDP-bound states, these mutants reveal striking
conformational peculiarities previously unobserved in other EF-Tu
structures.