Role of Dimerization in KH/RNA Complexes:  The Example of Nova KH3

Abstract

The K homology module, one of the most common RNA-binding motifs, is present in multiple copies in both prokaryotic and eukaryotic regulatory proteins. Increasing evidence suggests that self-aggregation of KH modules has a functional role. We have used a combination of techniques to characterize the behavior in solution of the third KH domain of Nova-1, a paradigmatic KH protein. The possibility of working on the isolated module allowed us to observe specifically the homodimerization and RNA-binding properties of KH domains. We provide conclusive evidence that self-association of Nova-1 KH3 occurs in solution even in the absence of RNA. Homodimerization involves a specific protein/protein interface. We also studied the dynamical behavior of Nova-1 KH3 in isolation and in complex with RNA. These data provide a model for the mechanism of KH/RNA recognition and suggest functional implications of dimerization in KH complexes. We discuss our findings in the context of the whole KH family and suggest a generalized mode of interaction.

The KH domains of Xenopus Vg1RBP mediate RNA binding and self-association

“…Vg1RBP can self-associate in vivo+ Mixed-stage Xenopus oocyte extract was crosslinked with buffer alone (Ϫ) or 0+3-1+5 mg/mL DMS+ Samples were resolved using SDS-PAGE alongside mock treated (Ϫ) or crosslinked (ϩ) samples of recombinant R-K12-K34 and subjected to western blot analysis using a-Vg1RBP antiserum+ The migration of Vg1RBP monomers, dimers, and the large complex is indicated on the right+ 1328 A. Git and N. Standart noncanonical RRMs, such as those in PTB (Conte et al+, 2000)+ At very high protein concentrations, the RRMs displayed a striking preference for poly(U) relative to poly(C), (A) and (G) (data not shown); though the physiological implications of this observation are not clear+ That the RRMs are dispensable for the RNA-binding activity of Vg1RBP agrees with their complete absence from the Drosophila homolog (Fig+ 1; Nielsen et al+, 2000), suggesting that they are required by the vertebrate homologs to support either binding to as yet unknown RNAs or additional functions, such as proteinprotein interactions+ Indeed, the RRMs appear to enhance K12 self-association (Fig+ 6)+ Finally, we also show that Vg1RBP self-associates, via the K34 didomain, in a manner that is greatly stabilized by RNA, both as recombinant protein, and in oocyte lysates (Figs+ 6 and 7)+ The notion that KH domains mediate oligomerization was first indirectly implied by the finding that KH3 from human Nova-1 antigen forms an intermolecular tetramer when crystallized (Lewis et al+, 1999)+ A recent NMR study of the Nova KH3 domain independently supports our findings that KH domains, although able to interact in the absence of RNA, are greatly stabilized in their protein-protein interactions by RNA+ Here, homodimerization involves a specific protein-protein interface and results in a general stiffening of the protein such that the protein binds to RNA by a rigidification of the protein/RNA-binding surface mediated by protein dimerization (Ramos et al+, 2002)+ However, the primary sequence of the Nova KH3 interacting surface is not conserved in Vg1RBP+…”

The role of a clinically important mutation in the fold and RNA-binding properties of KH motifs

“…In our previous work on the characterization of Nova-1 KH3 RNA-binding properties (Ramos et al 2002), we did not succeed in identifying the resonance of the amide proton of L447 in the NMR spectra, even when all distinguishable peaks had been assigned ( Fig. 2A).…”

“…Nova KH3 is one of the few examples for which the structures of both the isolated domain and of a KH/RNA complex are available (Lewis et al 1999(Lewis et al , 2000. Our choice was also suggested by the observation of an unusual behavior of L447 (the residue corresponding to I304 in FMRP) in the NMR spectrum of Nova-1 KH3: The resonance of the amide proton of L447 could not be identified (Ramos et al 2002). Here, we show that the apparently contradictory data on the function of the conserved residue stem from its dual role in protein stability and RNA recognition.…”

Distinct contributions of KH domains to substrate binding affinity of Drosophila P-element somatic inhibitor protein

“…The three KH domains of hnRNP K bind both ssDNA and RNA substrates, providing a very diverse functionality for this protein (Bomsztyk et al 2004). The KH domains of Nova-1 have been proposed to provide a surface for interor intramolecular self-association (Ramos et al 2002), and the KH3 domain of KSRP is responsible for interactions with the exosome (Gherzi et al 2004), implicating a role in protein-protein interactions for these domains as well.…”