The structural basis of tail-anchored membrane protein recognition by Get3.

Targeting of newly synthesized membrane proteins to the endoplasmic reticulum is an essential cellular process. Most membrane proteins are recognized and targeted co-translationally by the signal recognition particle. However, nearly 5% of membrane proteins are 'tail-anchored' by a single carboxy-terminal transmembrane domain that cannot access the co-translational pathway. Instead, tail-anchored proteins are targeted post-translationally by a conserved ATPase termed Get3. The mechanistic basis for tail-anchored protein recognition or targeting by Get3 is not known. Here we present crystal structures of yeast Get3 in 'open' (nucleotide-free) and 'closed' (ADP.AlF(4)(-)-bound) dimer states. In the closed state, the dimer interface of Get3 contains an enormous hydrophobic groove implicated by mutational analyses in tail-anchored protein binding. In the open state, Get3 undergoes a striking rearrangement that disrupts the groove and shields its hydrophobic surfaces. These data provide a molecular mechanism for nucleotide-regulated binding and release of tail-anchored proteins during their membrane targeting by Get3.

Structural rearrangements near the chromophore influence the maturation speed and brightness of DsRed variants.

The red fluorescent protein DsRed has been extensively engineered for use as an in vivo research tool. In fast maturing DsRed variants, the chromophore maturation half-time is approximately 40 min, compared to approximately 12 h for wild-type DsRed. Further, DsRed has been converted from a tetramer into a monomer, a task that entailed mutating approximately 20% of the amino acids. These engineered variants of DsRed have proven extremely valuable for biomedical research, but the structural basis for the improved characteristics has not been thoroughly investigated. Here we present a 1.7 A crystal structure of the fast maturing tetrameric variant DsRed.T4. We also present a biochemical characterization and 1.6 A crystal structure of the monomeric variant DsRed.M1, also known as DsRed-Monomer. Analysis of the crystal structures suggests that rearrangements of Ser69 and Glu215 contribute to fast maturation, and that positioning of the Lys70 side chain modulates fluorescence quantum yield. Despite the 45 mutations in DsRed.M1 relative to wild-type DsRed, there is a root-mean-square deviation of only 0.3 A between the two structures. We propose that novel intramolecular interactions in DsRed.M1 partially compensate for the loss of intermolecular interactions found in the tetramer.

A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing.

Group I introns often encode proteins that catalyze site-specific DNA hydrolysis. Some of these proteins have acquired the ability to promote splicing of their cognate intron, but whether these two activities reside in different regions of the protein remains obscure. A crystal structure of I-AniI, a dual function intron-encoded protein, has shown that the protein has two pseudo-symmetric domains of equal size. Each domain contacts its DNA substrate on either side of two cleavage sites. As a first step to identify the RNA binding surface, the N- and C-terminal domains of I-AniI were separately expressed and tested for promoting the splicing of the mitochondrial (mt) COB pre-RNA. The N-terminal protein showed no splicing activation or RNA binding, suggesting that this domain plays a minimal role in activity or is improperly folded. Remarkably, the 16-kDa C-terminal half facilitates intron splicing with a rate similar to that of the full-length protein. Both the C-terminal fragment and full-length proteins bind tightly to the COB intron. RNase footprinting shows that the C-terminal and full-length proteins bind to the same regions and induce the same conformational changes in the COB intron. Together, these results show that the C-terminal fragment of I-AniI is necessary and sufficient for maturase activity and suggests that I-AniI acquired splicing function by utilizing a relatively small protein surface that likely represents a novel RNA binding motif. This fragment of I-AniI represents the smallest group I intron splicing cofactor described to date.

Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor.

We determined the crystal structure of a bifunctional group I intron splicing factor and homing endonuclease, termed the I-AniI maturase, in complex with its DNA target at 2.6 A resolution. The structure demonstrates the remarkable structural conservation of the beta-sheet DNA-binding motif between highly divergent enzyme subfamilies. DNA recognition by I-AniI was further studied using nucleoside deletion and DMS modification interference analyses. Correlation of these results with the crystal structure provides information on the relative importance of individual nucleotide contacts for DNA recognition. Alignment and modeling of two homologous maturases reveals conserved basic surface residues, distant from the DNA-binding surface, that might be involved in RNA binding. A point mutation that introduces a single negative charge in this region uncouples the maturase and endonuclease functions of the protein, inhibiting RNA binding and splicing while maintaining DNA binding and cleavage.