ABSTRACT
NXF1-like members of the NXF (nuclear export factor) family orchestrate bulk nuclear export of mRNA, while functionally distinct NXF variant proteins carry out separate substrate-specific and tissue-specific RNA regulation. Metazoan organisms possess at least one NXF1-like gene and one or more NXF variant genes. Heterodimerization of both proteins with the NXT (NTF2-related export) protein is central to NXF family function; however, given the multiplicity of NXF/NXT complexes, the specificity and mechanism of heterodimerization remain unclear. Here, we report the structural and functional analyses of the Caenorhabditis elegans NXF variant ceNXF2 bound to ceNXT1. Contacts crucial for NXF/NXT heterodimer stability and specificity, including a probable site for phosphoregulation, have been identified. The ceNXF2 NTF2 domain bears at least two nucleoporin (Nup) binding pockets necessary for the colocalization of ceNXF2/ceNXT1 at the nuclear envelope. Unexpectedly, one Nup binding pocket is formed at the heterodimer interface of the ceNXF2/ceNXT1 complex, demonstrating that NXT binding directly regulates NXF function.
Subject(s)
Multiprotein Complexes/chemistry , Nucleocytoplasmic Transport Proteins/chemistry , Protein Folding , Protein Multimerization , RNA-Binding Proteins/chemistry , Amino Acid Sequence , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/metabolism , Humans , Models, Biological , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , Pliability , Protein Binding , Protein Structure, Quaternary , RNA-Binding Proteins/metabolism , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
Posttranscriptional regulation of gene expression is an important mechanism for modulating protein levels in eukaryotes, especially in developmental pathways. The highly conserved homodimeric STAR/GSG proteins play a key role in regulating translation by binding bipartite consensus sequences in the untranslated regions of target mRNAs, but the exact mechanism remains unknown. Structures of STAR protein RNA binding subdomains have been determined, but structural information is lacking for the homodimerization subdomain. Here, we present the structure of the C. elegans GLD-1 homodimerization domain dimer, determined by a combination of X-ray crystallography and NMR spectroscopy, revealing a helix-turn-helix monomeric fold with the two protomers stacked perpendicularly. Structure-based mutagenesis demonstrates that the dimer interface is not easily disrupted, but the structural integrity of the monomer is crucial for GLD-1 dimerization. Finally, an improved model for STAR-mediated translational regulation of mRNA, based on the GLD-1 homodimerization domain structure, is presented.
Subject(s)
Caenorhabditis elegans Proteins/chemistry , Amino Acid Sequence , Animals , Binding Sites , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Gene Expression Regulation , Molecular Sequence Data , Protein Biosynthesis , Protein Structure, Tertiary , RNA, Messenger/metabolism , RNA-Binding ProteinsABSTRACT
Only few instances are known of protein folds that tolerate massive sequence variation for the sake of binding diversity. The most extensively characterized is the immunoglobulin fold. We now add to this the C-type lectin (CLec) fold, as found in the major tropism determinant (Mtd), a retroelement-encoded receptor-binding protein of Bordetella bacteriophage. Variation in Mtd, with its approximately 10(13) possible sequences, enables phage adaptation to Bordetella spp. Mtd is an intertwined, pyramid-shaped trimer, with variable residues organized by its CLec fold into discrete receptor-binding sites. The CLec fold provides a highly static scaffold for combinatorial display of variable residues, probably reflecting a different evolutionary solution for balancing diversity against stability from that in the immunoglobulin fold. Mtd variants are biased toward the receptor pertactin, and there is evidence that the CLec fold is used broadly for sequence variation by related retroelements.
