Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: Conservation of the lateral RNA-binding mode

Abstract

SynopsisThe structure of an Hfq homolog from the deep-branching thermophilic bacterium Aquifex aeolicus, determined to 1.5-Å resolution both in apo form and bound to a uridine-rich RNA, reveals a conserved, pre-organized RNA-binding pocket on the lateral rim of the Hfq hexamer.AbstractThe host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNA) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings, with at least two distinct surfaces that bind RNA. Recently, another binding site—dubbed the ‘lateral rim’—has been implicated in sRNA•mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophile Aquifex aeolicus (Aae), but little is known about the structures and functions of Hfq from basal bacterial lineages such as the Aquificae. Thus, we have cloned, overexpressed, purified, crystallized, and biochemically characterized Aae Hfq. We have determined the structures of Aae Hfq in space-groups P1 and P6, both to 1.5 Å resolution, and we have discovered nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs. Co-crystallization with U6 RNA reveals that the outer rim of the Aae Hfq hexamer features a well-defined binding pocket that is selective for uracil. This Aae Hfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.

The Small β-Barrel Domain: A Survey-Based Structural Analysis

“…In some SBBs, these loops adopt known b-turn geometries (e.g., the b-turns described for Aquifex aeolicus Hfq; Stanek et al, 2017), while in other instances the loops are less regularly structured. Sheet A, also termed the Meander (Figure 1B), is a threestranded b-sheet consisting of strands b2C, b3, and b4 (contiguous in sequence and in space).…”

Structural biology meets data science: does anything change?

“…We believe that DS tools can address this need; note that texts are becoming available on this topic, such as the recent Big Data Visualization [42]. Ideas and methods from beyond SB-such as "chord diagram" layouts in genomics [43], termed "hierarchical edge bundles" [44] in computer graphics-can be applied in SB, for instance to visualize data associated with hierarchical clustering of protein structural differences (e.g., see the figures in [45]).…”

The Small β-barrel Domain: A Survey-based Structural Analysis

“…The strands in an SBB are generally rather short, being ≈4-6 residues long (see below), and are connected by loops; the loops in some SBBs adopt known β-turn geometries (see, e.g., β-turn geometries mentioned in A. aeolicus Hfq (Stanek et al 2017) ), while others are more irregularly structured. Sheet A, also referred to as the Meander (Fig 1B), is a three-stranded β-sheet consisting of strands β2C, β3 and β4 (contiguous in sequence and in space).…”

“…Structurally, one route seems to proceed through stacking of either the distal or proximal faces of two rings. SmAP rings have been shown to stack proximal -to-distal , while Hfq homologs have been found in proximal -to-distal (Stanek et al 2017) and distal -to-distal (Schumacher et al 2002) orientations. Certain geometric arrangements of Hfq and SmAP rings allow for runaway assembly into fibrillar polymers.…”

“…The helix stacks against the open barrel and, in the context of an intact, hexameric Hfq toroidal disc(Sauer 2013) , it lies atop the so-called proximal face (this face of the disc is defined below in Section 2.5.3; it has been termed the Loop L3 face for SmAPs(Mura et al 2013) ). In Hfq (b.38.1.2), the SBB's α-helix mediates interactions with cognate sRNA molecules via a patch of conserved basic residues (Arg16, Arg17, Arg19) and a Gln8 ((Schumacher et al 2002;Stanek et al 2017) ; note that E. coli Hfq residue numbering is used here). A similar mode of RNA-binding appears to be conserved in the Sm-like archaeal proteinsThore et al 2003) .…”