Refining the overall structure and subdomain orientation of ribosomal protein S4 Delta 41 with dipolar couplings measured by NMR in uniaxial liquid crystalline phases

Prokaryotic protein S4 initiates assembly of the small ribosomal subunit by binding to 16 S rRNA. Residues 43-200 of S4 from Bacillus stearothermophil us (S4 Delta 41) bind to both 16 S rRNA and to a mRNA pseudoknot. In order to obtain structure-based insights regarding RNA binding, we previously det ermined the solution structure of 54 Delta 41 using NOE, hydrogen bond, and torsion angle restraints. S4 Delta 41 is elongated, with two distinct: sub domains, one all helical, the other including a P-sheet. In contrast to the high resolution structures obtained for each individual subdomain, their r elative orientation was not precisely defined because only 17 intersubdomai n NOE restraints were determined. Compared to the 1.7 Angstrom crystal stru cture, when the sheet-containing subdomains are superimposed, the helical s ubdomain is twisted by almost 45 degrees about the long axis of the molecul e in the solution structure. Because variations in subdomain orientation ma y explain how the protein recognizes multiple RNA targets, our current goal is to determine the orientation of the subdomains in solution with high pr ecision. To this end, NOE assignments were re-examined. NOESY experiments o n a specifically labeled sample revealed that one of the intersubdomain res traints had been misassigned. However, the revised set of NOE restraints pr oduces solution structures that still have imprecisely defined subdomain or ientations and that lie between the original NMR structure and the crystal structure. In contrast, augmenting the NOE restraints with N-H dipolar coup lings, measured in uniaxial liquid crystalline phases, clearly establishes the relative orientation of the subdomains. Data obtained from two independ ent liquid crystalline milieux, DMPC/DHPC bicelles and the filamentous bact eriophage Pf1, show that the relative orientation of the subdomains in solu tion is quite similar to the subdomain orientation in the crystal structure . The solution structure, refined with dipolar data, is presented and its i mplications for S4's RNA binding activity are discussed. (C) 1999 Academic Press.