NMR in structural genomics to increase structural coverage of the protein universe: Delivered by Prof. Kurt Wüthrich on 7 July 2013 at the 38th FEBS Congress in St. Petersburg, Russia.

For more than a decade, the Joint Center for Structural Genomics (JCSG; www.jcsg.org) worked toward increased three-dimensional structure coverage of the protein universe. This coordinated quest was one of the main goals of the four high-throughput (HT) structure determination centers of the Protein Structure Initiative (PSI; www.nigms.nih.gov/Research/specificareas/PSI). To achieve the goals of the PSI, the JCSG made use of the complementarity of structure determination by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy to increase and diversify the range of targets entering the HT structure determination pipeline. The overall strategy, for both techniques, was to determine atomic resolution structures for representatives of large protein families, as defined by the Pfam database, which had no structural coverage and could make significant contributions to biological and biomedical research. Furthermore, the experimental structures could be leveraged by homology modeling to further expand the structural coverage of the protein universe and increase biological insights. Here, we describe what could be achieved by this structural genomics approach, using as an illustration the contributions from 20 NMR structure determinations out of a total of 98 JCSG NMR structures, which were selected because they are the first three-dimensional structure representations of the respective Pfam protein families. The information from this small sample is representative for the overall results from crystal and NMR structure determination in the JCSG. There are five new folds, which were classified as domains of unknown functions (DUF), three of the proteins could be functionally annotated based on three-dimensional structure similarity with previously characterized proteins, and 12 proteins showed only limited similarity with previous deposits in the Protein Data Bank (PDB) and were classified as DUFs.

Directional Phosphorylation and Nuclear Transport of the Splicing Factor SRSF1 Is Regulated by an RNA Recognition Motif.

Multisite phosphorylation is required for the biological function of serine-arginine (SR) proteins, a family of essential regulators of mRNA splicing. These modifications are catalyzed by serine-arginine protein kinases (SRPKs) that phosphorylate numerous serines in arginine-serine-rich (RS) domains of SR proteins using a directional, C-to-N-terminal mechanism. The present studies explore how SRPKs govern this highly biased phosphorylation reaction and investigate biological roles of the observed directional phosphorylation mechanism. Using NMR spectroscopy with two separately expressed domains of SRSF1, we showed that several residues in the RNA-binding motif 2 interact with the N-terminal region of the RS domain (RS1). These contacts provide a structural framework that balances the activities of SRPK1 and the protein phosphatase PP1, thereby regulating the phosphoryl content of the RS domain. Disruption of the implicated intramolecular RNA-binding motif 2-RS domain interaction impairs both the directional phosphorylation mechanism and the nuclear translocation of SRSF1 demonstrating that the intrinsic phosphorylation bias is obligatory for SR protein biological function.

Chirality and template-mediated induction of helical preferences in achiral ß-peptides.

This study describes chirality- or template-mediated helical induction in achiral ß-peptides for the first time. A strategy of end capping ß-peptides derived from ß-hGly (the smallest achiral ß-amino acid) with a chiral ß-amino acid that possesses a carbohydrate side chain (ß-Caa; C-linked carbo ß-amino acid) or a small, robust helical template derived from ß-Caas, was adopted to investigate folding propensity. A single chiral (R)-ß-Caa residue at the C- or N-terminus in these oligomers led to a preponderance of right-handed 12/10-helical folds, which was reiterated more strongly in peptides capped at both the C- and N-terminus. Likewise, the presence of a template (a 12/10-helical trimer) at both the C- and N-terminus resulted in a very robust helix. The propagation of the helical fold and its sustenance was found in a homo-oligomeric sequence with as many as seven ß-hGly residues. In both cases, the induction of helicity was stronger from the N terminus, whereas an anchor at the C terminus resulted in reduced helical propensity. Although these oligomers have been theoretically predicted to favor a 12/10-mixed helix in apolar solvents, this study provides the first experimental evidence for their existence. Diastereotopicity was found in both the methylene groups of the ß-hGly moieties due to chirality. Additionally, the ß-hGly units have shown split behavior in the conformational space to accommodate the 12/10-helix. Thus, end capping to assist chiralty- or template-mediated helical induction and stabilization in achiral ß-peptides is a very attractive strategy.

Design of a "new motif" with beta-amino acids and alpha-aminoxy acids: synthesis of hybrid peptides with 12/10-helix.

Hybrid peptides are prepared from a C-linked carbo-beta-amino acid ester (R-beta-Caa) and an alpha-aminoxy acid (R-Ama) derived from S-lactic acid. Extensive NMR (in CDCl 3 solution), CD, and MD studies on the tetra- and hexapeptides led to identification of robust 12/10-mixed helices. The dipeptide repeat having an R-beta-Caa and an R-Ama thus provides a "new motif" to realize a 12/10-mixed helix, for the first time, in oligomers containing R-Ama. To understand the impact of side chains in the mixed helix formation, R-beta-Caa/Ama (with no substitution in Ama) and S-beta-hAla/R-Ama oligomers were investigated. NMR studies revealed the existence of 12/10-helices in these hybrid peptides, and the side chains of monomers were found to have a profound influence on their stabilities. These observations imply that the propensity of beta-amino acid to prefer a mixed 12/10-helix governs the structural behavior in these peptides. The structural consequences of the lone-pair repulsion between nitrogen and oxygen atoms result in a new and interesting structural motif which behaves like "pseudo" beta (3),beta(2)-peptides in generating 12/10-mixed helices.