Dynamic Requirements for a Functional Protein Hinge

• Published in: Journal of molecular biology 2007-04, Vol.368 (1), p.131-149
• Main Authors: Kempf, James G., Jung, Ju-yeon, Ragain, Christina, Sampson, Nicole S., Loria, J. Patrick
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Catalytic Domain; Chickens; enzyme catalysis; Glycine - genetics; loop motion; Models, Biological; Models, Molecular; Mutant Proteins - chemistry; Mutation; NMR spectroscopy; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; protein dynamics; Protein Folding; protein hinge; Protein Structure, Tertiary; Triose-Phosphate Isomerase - chemistry; Triose-Phosphate Isomerase - genetics; Triose-Phosphate Isomerase - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2007.01.074

The enzyme triosephosphate isomerase (TIM) is a model of catalytic efficiency. The 11 residue loop 6 at the TIM active site plays a major role in this enzymatic prowess. The loop moves between open and closed states, which facilitate substrate access and catalysis, respectively. The N and C-terminal hinges of loop 6 control this motion. Here, we detail flexibility requirements for hinges in a comparative solution NMR study of wild-type (WT) TIM and a quintuple mutant (PGG/GGG). The latter contained glycine substitutions in the N-terminal hinge at Val167 and Trp168, which follow the essential Pro166, and in the C-terminal hinge at Lys174, Thr175, and Ala176. Previous work demonstrated that PGG/GGG has a tenfold higher K m value and 10 3-fold reduced k cat relative to WT with either d-glyceraldehyde 3-phosphate or dihyrdroxyacetone phosphate as substrate. Our NMR results explain this in terms of altered loop-6 dynamics in PGG/GGG. In the mutant, loop 6 exhibits conformational heterogeneity with corresponding motional rates < 750 s − 1 that are an order of magnitude slower than the natural WT loop 6 motion. At the same time, nanosecond timescale motions of loop 6 are greatly enhanced in the mutant relative to WT. These differences from WT behavior occur in both apo PGG/GGG and in the form bound to the reaction-intermediate analog, 2-phosphoglycolate (2-PGA). In addition, as indicated by 1H, 15N and 13CO chemical-shifts, the glycine substitutions diminished the enzyme's response to ligand, and induced structural perturbations in apo and 2-PGA-bound forms of TIM that are atypical of WT. These data show that PGG/GGG exists in multiple conformations that are not fully competent for ligand binding or catalysis. These experiments elucidate an important principle of catalytic hinge design in proteins: structural rigidity is essential for focused motional freedom of active-site loops.