Alteration of Hydrogen Bonding in the Vicinity of Histidine 48 Disrupts Millisecond Motions in RNase A

Abstract

The motion of amino acid residues on the millisecond (ms) time scale is involved in the tight regulation of catalytic function in numerous enzyme systems. Using a combination of mutational, enzymological, and relaxation-compensated (15)N Carr-Purcell-Meiboom-Gill (CPMG) methods, we have previously established the conformational significance of the distant His48 residue and the neighboring loop 1 in RNase A function. These studies suggested that RNase A relies on an intricate network of hydrogen bonding interactions involved in propagating functionally relevant, long-range ms motions to the catalytic site of the enzyme. To further investigate the dynamic importance of this H-bonding network, this study focuses on the individual replacement of Thr17 and Thr82 with alanine, effectively altering the key H-bonding interactions that connect loop 1 and His48 to the rest of the protein. (15)N CPMG dispersion studies, nuclear magnetic resonance (NMR) chemical shift analysis, and NMR line shape analysis of point mutants T17A and T82A demonstrate that the evolutionarily conserved single H-bond linking His48 to Thr82 is essential for propagating ms motions from His48 to the active site of RNase A on the time scale of catalytic turnover, whereas the T17A mutation increases the off rate and conformational exchange motions in loop 1. Accumulating evidence from our mutational studies indicates that residues experiencing conformational exchange in RNase A can be grouped into two separate clusters displaying distinct dynamical features, which appear to be independently affected by mutation. Overall, this study illuminates how tightly controlled and finely tuned ms motions are in RNase A, suggesting that designed modulation of protein motions may be possible.

Conservation of Flexible Residue Clusters among Structural and Functional Enzyme Homologues

“…Interestingly, cluster 3 in RNase A encompasses the same long scale residues experiencing chemical shift variations upon pyrimidine binding (Fig. 2b), in line with previous hypotheses linking motions of this cluster to product release in RNase A (22)(23)(24)(25). However, efforts to correlate free and 3Ј-UMP-or 5Ј-AMP-bound 15 N chemical shift variations (⌬␦ ppm) with 15 N chemical shift differences between the major and the minor excited state obtained from 15 N CPMG (⌬) remained inconclusive (supplemental Fig.…”

“…3) , Gln 101 , all part of cluster 3) also show important conformational exchange on the millisecond time scale and are known to be involved in the propagation of motions between loop 1 and the active site during turnover (22,23). Coupled to the chemical shift differences induced by 3Ј-UMP binding, this observation strongly suggests that long scale millisecond motions connecting the active site to loop 1 (cluster 3) are involved in the binding/release of pyrimidine ligands in RNase A.…”

“…(also located in ␤1 and ␤4), providing important pyrimidine interactions with RNA ligands during catalysis (23,24). As was demonstrated by mutagenesis (22,23,25) and unnatural amino acid modifications (24), this concerted dynamic network is essential for optimal catalysis in RNase A, acting by modulating the rate-limiting step of the reaction (product release (18).…”

“…We and others have shown that RNase A requires concerted millisecond (ms) dynamics for efficient catalysis through motions involving a distant loop (loop 1, residues 14 -26) located more than 20 Å away from the reactive center (22). During turnover, the flexible loop 1 propagates ms motions to the active site through a highly conserved pair of hydrogen-bonded residues (His 48 -Thr 82 ), which are located on the adjacent ␤1 and ␤4 strands (23). These molecular motions are transmitted over a length scale of more than 20 Å from loop 1 to active site residues Thr 45 and Asp…”

“…As was demonstrated by mutagenesis (22,23,25) and unnatural amino acid modifications (24), this concerted dynamic network is essential for optimal catalysis in RNase A, acting by modulating the rate-limiting step of the reaction (product release (18). Based on these observations, it is tempting to verify whether evolutionarily conserved motional networks between homologous ribonucleases could partially account for their divergent catalytic activities.…”