Crystal structure of the U1A spliceosomal protein complexed with its cognate RNA hairpin.

We have determined the crystal structure of the RNA binding domain of the U1A spliceosomal protein bound to a 21-nucleotide RNA hairpin at 1.92 A resolution. The ten-nucleotide RNA loop binds to the surface of the four-stranded beta-sheet of the RNP domain as an open structure. The AUUGCAC hexanucleotide sequence interacts extensively with the conserved RNP1 and RNP2 motifs and the C-terminal extension of the RNP domain. The stacking interaction between RNA bases and aromatic protein side chains and the extensive hydrogen bonding network involving RNA bases, protein side chains and protein mainchain amide and carbonyl groups are crucial for the sequence specific recognition of RNA.

Mutant hemoglobins (alpha 119-Ala and beta 55-Ser): functions related to high-altitude respiration in geese.

The unusually high blood-O2 affinity in the bar-headed and Andean geese is a necessary adaptation for migration across high mountain ranges. The amino acid residues alpha-119 and beta-55, which form an alpha 1 beta 1 contact in human hemoglobin (Hb), are altered in bar-headed and Andean geese, respectively, which suggests that loss of this contact increases O2 affinity. Two mutant human Hbs with equivalent mutations at these sites prepared by site-directed mutagenesis show the same increase in O2 affinity compared with Hb A, which indicates that these mutations are responsible for the changes in the protein. The intrinsic affinity difference compared with native Hb A is amplified by organic phosphates. Whereas the recombinant and native Hbs displayed similar sensitivities to pH, chloride, and 2,3-diphosphoglycerate, the oxygenation heat of the alpha-chain mutant decreased in the presence of 2,3-diphosphoglycerate. O2 association constants for the deoxygenated state of the alpha-mutant were about three times those for Hb A. The mutant Hb analogously exhibited higher affinity constants for binding the first three O2 molecules. Calculated heme-heme interaction energies indicated that loss of a single contact, resulting in destabilization of the deoxy (tense) structure, underlies the increased O2 affinity. Adaptations securing Hb-O2 binding at extreme altitude are discussed.

Identification of molecular contacts between the U1 A small nuclear ribonucleoprotein and U1 RNA.

We recently determined the crystal structure of the RNP domain of the U1 small nuclear ribonucleoprotein A and identified Arg and Lys residues involved in U1 RNA binding. These residues are clustered around the two highly conserved segments, RNP1 and RNP2, located in the central two beta strands. We have now studied the U1 RNA binding of mutants where potentially hydrogen bonding residues on the RNA binding surface were replaced by non-hydrogen bonding residues. In the RNP2 segment, the Thr11----Val and Asn15----Val mutations completely abolished, and the Tyr13----Phe and Asn16----Val mutations substantially reduced the U1 RNA binding, suggesting that these residues form hydrogen bonds with the RNA. In the RNP1 segment Arg52----Gln abolished, but Arg52----Lys only slightly affected U1 RNA binding, suggesting that Arg52 may form a salt bridge with phosphates of U1 RNA. Ethylation protection experiments of U1 RNA show that the backbone phosphates of the 3' two-thirds of loop II and the 5' stem are in contact with the U1 A protein. The U1 A protein-U1 RNA binding constant is substantially reduced by A----G and G----A replacements in loop II, but not by C----U or U----C replacements. Based on these biochemical data we propose a structure for the complex between the U1 A ribonucleoprotein and U1 RNA.

Adaptation of bird hemoglobins to high altitudes: demonstration of molecular mechanism by protein engineering.

Of two closely related species of geese, one, the greylag goose, lives in the Indian plains all year round, while the other, the bar-headed goose, lives at the Tibetan lakes and migrates across the Himalayas to winter in India. Another species, the Andean goose, lives in the High Andes all year round. Possession of a Hb with high oxygen affinity helps to adapt bar-headed and Andean geese to high altitudes. The Hb amino acid sequences of the bar-headed and the greylag geese differ by four substitutions, of which only one is unique among bird sequences: Pro-119 alpha (H2)----Ala. Perutz proposed that the two-carbon gap left by this substitution at the alpha 1 beta 1 contact raises the oxygen affinity, because it relaxes the tension in the deoxy or T structure [Perutz, M. F. (1983) Mol. Biol. Evol. 1, 1-28]. It was later found that the Hb of the Andean goose has a gap in the same position, due to the complementary substitution Leu-55 beta (D6)----Ser. We have tested Perutz's hypothesis by introducing each of these substitutions into human globin synthesized in Escherichia coli. The reconstituted Hbs combine cooperatively with oxygen. Their oxygen affinities exceed that of normal human Hb by an even larger factor than that found between the high-flying geese and the greylag goose. The mutant Hb Met-55 beta (D6)----Ser was crystallized. Its structure is the same as that of HbA, except in the immediate environment of the gap left by the substitution of the serine for the methionine side chain, which evidently causes the increased oxygen affinity of this Hb.

Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A.

The crystal structure of the RNA binding domain of the U1 small nuclear ribonucleoprotein A, which forms part of the ribonucleoprotein complex involved in the excision of introns, has been solved. It contains a four-stranded beta sheet and two alpha helices. The highly conserved segments designated RNP1 and RNP2 lie side by side on the middle two beta strands. U1 RNA binding studies of mutant proteins suggest that the RNA binds to the four-stranded beta sheet and to the flexible loops on one end.