Volume 7, Issue 6, Pages 1177-1189 (June 2001) An Extended RNA Binding Surface through Arrayed S1 and KH Domains in Transcription Factor NusA  Michael Worbs, Gleb P. Bourenkov, Hans D. Bartunik, Robert Huber, Markus C. Wahl  Molecular Cell  Volume 7, Issue 6, Pages 1177-1189 (June 2001) DOI: 10.1016/S1097-2765(01)00262-3

Figure 1 Overall Structure (a) Exemplary section of an experimental MAD map at 2.8 Å resolution. (b) Stereo ribbon diagram of tmaNusA. Secondary structure elements and domains are indicated. (c) The left-hand side shows the putative RNA binding surface with positive electrostatic potential. Yellow ribbons mark RNA ligands for the S1 and KH motifs. The right-hand side shows the surface opposite the positively charged stretch. (d) nusA1 (I182R in tmaNusA) and nusA11 (V180D in tmaNusA) mutations mapped to the S1 hydrophobic core. The domain is in the same orientation as in (b). (e) Map of NusA mutations (large spheres) discussed in the text. “449” marks the so-called 449 region (see text). Other mutations are labeled by residue numbers Molecular Cell 2001 7, 1177-1189DOI: (10.1016/S1097-2765(01)00262-3)

Figure 2 Sequence Alignment Alignment of bacterial NusA sequences (Thermotoga maritima, Escherichia coli, Haemophilus influenzae, Borrelia burgdorferi, Thermus thermophilus, Mycoplasma genitalium, and Bacillus subtilis). Numbering corresponds to tmaNusA. The background of amino acids identical in at least six of the seven species is colored red, and those with five conserved sequences are yellow. The secondary structure is colored according to the domains (bottom). Black triangles mark residues that are part of the positive flank Molecular Cell 2001 7, 1177-1189DOI: (10.1016/S1097-2765(01)00262-3)

Figure 3 RNA Binding Models (a) Overlay of the NusA S1 domain (red) with the N-terminal OB fold motif of Asp-tRNA synthetase (blue) in complex with the tRNA anticodon loop (gold) (Eiler et al., 1999). The view is from the upper left corner of Figure 1b, with the NTD at the bottom and KH1 at the top. (b) Overlay of the first KH domain of NusA (red) and the NOVA KH3 domain (blue) in complex with a RNA hairpin (gold) (Lewis et al., 2000). The view is from the direction of NusA KH2 (bottom of Figure 1b) onto the KH1 β sheet. In both panels, known and putative RNA-contacting peptides are colored cyan and pink for Asp-tRNA synthetase/NOVA and NusA sequences, respectively. GXXG, GXXG turn of the helix-turn-helix motif; VL, variable loop (NOVA); AL, additional loop (NusA). (c) The different topologies of eukaryotic and bacterial KH domains. Green denotes a common core motif, and gray denotes terminal extensions. Yellow stars indicate RNA contact sites Molecular Cell 2001 7, 1177-1189DOI: (10.1016/S1097-2765(01)00262-3)

Figure 4 Domain Stacking (a) Closeup of the interactions between S1 and KH1. The view is from the back of Figure 1b, with S1 at the left and KH1 at the right. (b) Details of the KH1–KH2 contacts. The view is from the left in Figure 1b, with KH1 rotated to the left and KH2 to the right. Absolutely conserved residues involved in the interactions are labeled Molecular Cell 2001 7, 1177-1189DOI: (10.1016/S1097-2765(01)00262-3)

Figure 5 KH Arrays (a) Arrangement of the NusA KH domains via opposite poles. The view is from the bottom right of Figure 1b. (b) Ribbon diagram of the same region with a KH1-bound RNA hairpin rotated ∼90° counterclockwise about the longitudinal axis. The prime RNA binding peptides as deduced from Figure 3 are labeled (red). Additional contacts through KH2 are in green. (c) Crystal contacts of two NOVA KH3·RNA complexes. The bottom KH domains in (a) and (c) are in approximately the same orientation Molecular Cell 2001 7, 1177-1189DOI: (10.1016/S1097-2765(01)00262-3)

Figure 6 Protein Contacts Residues involved in protein contacts in other OB fold and KH domain proteins from the Protein DataBank mapped in green on the S1/KH region of NusA. RNAs are shown as yellow ribbons. Part of the KH1 RNA binding site appears as a protein interaction face only because of its intimate contact to KH2 in NusA, a consequence of the cooperation between the modules (see text). Consistently, the equivalent surface in KH2 is not labeled. Views are identical to those of Figure 1c Molecular Cell 2001 7, 1177-1189DOI: (10.1016/S1097-2765(01)00262-3)