The crystal structure of the signal recognition particle Alu RNA binding heterodimer, SRP9/14

Abstract

The mammalian signal recognition particle (SRP) is an 11S cytoplasmic ribonucleoprotein that plays an essential role in protein sorting. SRP recognizes the signal sequence of the nascent polypeptide chain emerging from the ribosome, and targets the ribosome–nascent chain–SRP complex to the rough endoplasmic reticulum. The SRP consists of six polypeptides (SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72) and a single 300 nucleotide RNA molecule. SRP9 and SRP14 proteins form a heterodimer that binds to the Alu domain of SRP RNA which is responsible for translation arrest. We report the first crystal structure of a mammalian SRP protein, that of the mouse SRP9/14 heterodimer, determined at 2.5 Å resolution. SRP9 and SRP14 are found to be structurally homologous, containing the same α‐β‐β‐β‐α fold. This we designate the Alu binding module (Alu bm), an additional member of the family of small α/β RNA binding domains. The heterodimer has pseudo 2‐fold symmetry and is saddle like, comprising a strongly curved six‐stranded amphipathic β‐sheet with the four helices packed on the convex side and the exposed concave surface being lined with positively charged residues.

Hierarchical assembly of the Alu domain of the mammalian signal recognition particle

“…The signal recognition particle (SRP; Walter & Blobel, 1980), like the ribosome, is a cytoplasmic ribonucleoprotein particle (RNP) of ancient evolutionary origin (Poritz et al+, 1990;Bhuiyan et al+, 2000)+ SRP has an intrinsic affinity for ribosomes (Walter et al+, 1981) and its catalytic promotion of the cotranslational mode of protein translocation across membranes is well documented (Walter & Johnson, 1994;Lütcke, 1995)+ In mammals, SRP consists of the highly base-paired 300-nt-long SRP RNA and six proteins: SRP54, SRP19, and the heterodimers SRP68/72 and SRP9/14 (Fig+ 1)+ SRP9/14 associates with the terminal sequences of SRP RNA, forming the enzymatically separable Alu domain of SRP (Gundelfinger et al+, 1983), whereas the other proteins together with the central RNA sequence form the S-domain of SRP+ High resolution crystal structures are now available for a number of SRP components: the NG-and M-domains of SRP54 (Freymann et al+, 1997;Montoya et al+, 1997;Keenan et al+, 1998;Clemons et al+, 1999) and the M-domain in complex with helix 8 of SRP RNA (Batey et al+, 2000) as well as the free helices 6 (Wild et al+, 1999) and 8 (Jovine et al+, 2000) of SRP RNA, free SRP9/14 (Birse et al+, 1997), and, most recently, the Alu domain with SRP9/14 clamping together in its concave beta-sheet the 59 and 39 domains of Alu RNA (Weichenrieder et al+, 2000)+ In electron micrographs the particle appears as a flexible, tri-segmented rod of 60 Å by 260-280 Å with the two domains distinguishable at opposite ends (Andrews et al+, 1985(Andrews et al+, , 1987+ SRP selects ribosomes displaying the N-terminal signal sequence of nascent secretory and membrane proteins that first emerges at the exit pore on the large ribosomal subunit+ The Alu domain of SRP is responsible for retarding the elongation of these proteins once their export signal sequence is bound by the S-domain of SRP and prior to engagement with the translocation machinery in the endoplasmic reticulum+ Considering the apparent length of the particle, it has been pro-posed that the Alu domain might reach the cleft between the two ribosomal subunits where the elongation factors bind (Andrews et al+, 1987;Siegel & Walter, 1988), but the Alu RNP crystal structure does not support the hypothesis that elongation arrest is caused by a mechanism of mimicry-based active competition with elongation factors (Weichenrieder et al+, 2000)+ Knowledge of the preferred orientation and the degree of flexibility of the Alu domain (and the crucial C-termi...…”

New Insights into Signal Recognition and Elongation Arrest Activities of the Signal Recognition Particle

“…Accession numbers: Arabidopsis thaliana Y10116, Oryza sativa Y10118, Mus musculus M29264, Homo sapiens X73459, Saccharomyces cerevisiae L35155. acids 90 -110 in SRP14 of which G93 and K95 make contact with SRP9 in the heterodimer (Birse et al, 1997) decreased not only the stability of the heterodimer as expected, but also resulted in a 10 fold reduced stability of the RNA-protein complex (Thomas et al, 1997). Using hydroxyl radicals as a tertiary structure probe, conformation- Two different orientations (A) and (B) are given, which are perpendicular to each other.…”

“…In analogy to the U1A snRNP and the MS2 proteins (Oubridge et al, 1994;Valegard et al, 1994), these findings suggested that the ␤-sheet surface may be involved in contacting RNA (Birse et al, 1997). The biochemical analysis of mutated SRP14 and SRP9 proteins identified two regions that are critical for RNA-binding.…”

“…The crystal structure of the proteins revealed that they belong to the family of small ␣/␤ RNA binding proteins and that, despite the absence of noticeable primary sequence homology, they have an almost identical ␣-␤-␤-␤-␣ fold (Birse et al, 1997). Together, they form a six-stranded antiparallel ␤-sheet stacked against four ␣-helices with a pseudo two-fold symmetry (Figure 3 and 4).…”

“…In this model, the pseudoknot structure is stabilised when the protein is bound. The experimentally derived 3D coordinates of SRP9/14 were taken from Birse et al (1997). A model of the RNA pseudoknot is provided by Zwieb et al, 1996 (http://pegasus.uthct.edu/SRPDB/SRPDB).…”