Exploring protein interiors: The role of a buried histidine in the KH module fold

The K-homology (RH) module is a novel RNA-binding motif. The structures of a representative KH motif from vigilin (vig-KH6) and of the first HH domain of fmr1 have been recently solved by nuclear magnetic resonance (NMR) and automated assignment-refinement techniques (ARIA). While a hydrophobic resi due is found at position 21 in most of the RH modules, a buried His is cons erved in all the 15 KH repeats of vigilin, This position must therefore hav e a key structural role in stabilizing the hydrophobic core. In Me present work, we have addressed Me following questions in order to obtain a detaile d description of the role of His 21: i) what is the exact role of the histi dine in the hydrophobic core of vig-KH6? ii) can we define the interactions that allow a conserved buried position to be occupied by a histidine both in vig-KH6 and in the whole vigilin HH sub-family? iii) how is the structur e and stability of vig-KH6 influenced by the state of protonation of this h istidine? To answer these questions, we have carried out an extensive refin ement of the vig-KH6 structure using both an improved ARIA protocol startin g from different initial structures and successively running restrained and unrestrained trajectories in water. An analysis of the stability of second ary structural elements, solvent accessibility, and hydrogen bonding patter ns allows hypothesis on Me structural role of residue His 21 and on the int eractions that this residue forms with the environment, The importance of t he protonation state of His 21 on Me stability of the KH fold was addressed and validated by experimental results. Proteins 1999;34:484-496. (C) 1999 Wiley-Liss, Inc.