Lu-Yun Lian

Molecular basis of RNA recognition and TAP binding by the SR proteins SRp20 and 9G8

The sequence‐specific RNA‐binding proteins SRp20 and 9G8 are the smallest members of the serine‐ and arginine‐rich (SR) protein family, well known for their role in splicing. They also play a role in mRNA export, in particular of histone mRNAs. We present the solution structures of the free 9G8 and SRp20 RNA recognition motifs (RRMs) and of SRp20 RRM in complex with the RNA sequence 5′CAUC3′. The SRp20‐RNA structure reveals that although all 4 nt are contacted by the RRM, only the 5′ cytosine is primarily recognized in a specific way. This might explain the numerous consensus sequences found by SELEX (systematic evolution of ligands by exponential enrichment) for the RRM of 9G8 and SRp20. Furthermore, we identify a short arginine‐rich peptide adjacent to the SRp20 and 9G8 RRMs, which does not contact RNA but is necessary and sufficient for interaction with the export factor Tip‐associated protein (TAP). Together, these results provide a molecular description for mRNA and TAP recognition by SRp20 and 9G8.

Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP

Adaptor proteins stimulate the nuclear export of mRNA, but their mechanism of action remains unclear. Here, we show that REF/ALY binds mRNA; but upon formation of a ternary complex with TAP the RNA is transferred from REF to TAP, and overexpression of TAP displaces REF from mRNA in vivo. RNA is also handed over from two other adaptors, 9G8 and SRp20 to TAP upon formation of a ternary complex. Interestingly, the RNA-binding affinity of TAP is enhanced 4-fold in vitro once it is complexed with REF. 9G8 and SRp20 also enhance the TAP RNA-binding activity in vitro. Consistent with a model in which TAP directly binds mRNA handed over from adaptors during export, we show that TAP binds mRNA in vivo by an arginine-rich motif in its N-terminal domain. The importance of direct TAP–mRNA interactions is confirmed by the observation that a mutant form of TAP that fails to bind mRNA but retains the ability to bind REF does not function in mRNA export.

A modulator of rho family G proteins, rhoGDI, binds these G proteins via an immunoglobulin-like domain and a flexible N-terminal arm

BACKGROUND The rho family of small G proteins, including rho, rac and cdc42, are involved in many cellular processes, including cell transformation by ras and the organization of the actin cytoskeleton. Additionally, rac has a role in the regulation of phagocyte NADPH oxidase. Guanine nucleotide dissociation inhibitors (GDIs) of the rhoGDI family bind to these G proteins and regulate their activity by preventing nucleotide dissociation and by controlling their interaction with membranes. RESULTS We report the structure of rhoGDI, determined by a combination of X-ray crystallography and NMR spectroscopy. NMR spectroscopy and selective proteolysis show that the N-terminal 50-60 residues of rhoGDI are flexible and unstructured in solution. The 2.5 A crystal structure of the folded core of rhoGDI, comprising residues 59-204, shows it to have an immunoglobulin-like fold, with an unprecedented insertion of two short beta strands and a 310 helix. There is an unusual pocket between the beta sheets of the immunoglobulin fold which may bind the C-terminal isoprenyl group of rac. NMR spectroscopy shows that the N-terminal arm is necessary for binding rac, although it remains largely flexible even in the complex. CONCLUSIONS The rhoGDI structure is notable for the existence of both a structured and a highly flexible domain, both of which appear to be required for the interaction with rac. The immunoglobulin-like fold of the structured domain is unusual for a cytoplasmic protein. The presence of equivalent cleavage sites in rhoGDI and the closely related D4/Ly-GDI (rhoGDI-2) suggest that proteolytic cleavage between the flexible and structured regions of rhoGDI may have a role in the regulation of the activity of members of this family. There is no detectable similarity between the structure of rhoGDI and the recently reported structure of rabGDI, which performs the same function as rhoGDI for the rab family of small G proteins.

Structure‐activity relationships in the peptide antibiotic nisin: antibacterial activity of fragments of nisin

The post‐translationally modified peptide antibiotic nisin has been cleaved by a number of proteases and the fragments produced purified, characterised chemically, and assayed for activity in inhibiting the growth of Lactococcus lactis MG1614 and Micrococcus luteus NCDO8166. These results provide information on the importance of different parts of the nisin molecule for its growth‐inhibition activity. Removal of the C‐terminal five residues leads to approximately a 10‐fold decrease in potency, while removal of a further nine residues, encompassing two of the lanthionine rings, leads to a 100‐fold decrease. There are some differences between analogous fragments of nisin and subtilin, suggesting possible subtle differences in mode of action. Cleavage within, or removal of, lanthionine ring C essentially abolishes the activity of nisin. The fragment nisin1−12 is inactive itself, and specifically antagonises the growth‐inhibitory action of nisin. These results are discussed in terms of current models for the mechanism of action of nisin.

Isolation and characterisation of two degradation products derived from the peptide antibiotic nisin

Two degradation products of nisin have been isolated and their structures have been determined by 1H NMR. Nisin1–32 [(des‐ΔAla33‐Lys34; Val32‐NH2)nisin] and (des‐ΔAla5)nisin1–32 [(des‐ΔAla5, ΔAla33‐Lys34; Ile4‐NH2, pyruvyl‐Leu6, Val32‐NH2)nisin] are formed on storage or by acid treatment. Contrary to previous reports, nisin1–32 showed potent antimicrobial activity against Gram‐positive organisms comparable to that of nisin itself. (des‐ΔAla5)Nisin1–32, however, was biologically inactive, thus demonstrating the importance of ΔAla5 and/or ring A for biological activity.

ParG, a protein required for active partition of bacterial plasmids, has a dimeric ribbon-helix-helix structure.

The ParG protein (8.6 kDa) is an essential component of the DNA partition complex of multidrug resistance plasmid TP228. ParG is a dimer in solution, interacts with DNA sequences upstream of the parFG genes and also with the ParF partition protein both in the absence and presence of target DNA. Here, the solution nuclear magnetic resonance structure of ParG is reported. The ParG dimer is composed of a folded domain formed by two closely intertwined C‐terminal parts (residues 33–76), and two highly mobile tails consisting of N‐terminal regions (residues 1–32). The folded part of ParG has the ribbon–helix–helix (RHH) architecture similar to that of the Arc/MetJ superfamily of DNA‐binding transcriptional repressors, although the primary sequence similarity is very low. ParG interacts with DNA predominantly via its folded domain; this interaction is coupled with ParG oligomerization. The dimeric RHH structure of ParG suggests that it binds to DNA by inserting the double‐stranded β‐sheet into the major groove of DNA, in a manner similar to transcriptional repressors from the Arc/MetJ superfamily, and that ParG can function as a transcriptional repressor itself. A new classification of proteins belonging to the Arc/MetJ superfamily and ParG homologues is proposed, based on the location of a conserved positively charged residue at either the beginning or at the end of the β‐strand which forms part of the DNA recognition motif.

Toxin–antitoxin regulation: bimodal interaction of YefM–YoeB with paired DNA palindromes exerts transcriptional autorepression

Toxin–antitoxin (TA) complexes function in programmed cell death or stress response mechanisms in bacteria. The YefM–YoeB TA complex of Escherichia coli consists of YoeB toxin that is counteracted by YefM antitoxin. When liberated from the complex, YoeB acts as an endoribonuclease, preferentially cleaving 3′ of purine nucleotides. Here we demonstrate that yefM-yoeB is transcriptionally autoregulated. YefM, a dimeric protein with extensive secondary structure revealed by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, is the primary repressor, whereas YoeB is a repression enhancer. The operator site 5′ of yefM-yoeB comprises adjacent long and short palindromes with core 5′-TGTACA-3′ motifs. YefM binds the long palindrome, followed sequentially by short palindrome recognition. In contrast, the repressor–corepressor complex recognizes both motifs more avidly, impyling that YefM within the complex has an enhanced DNA-binding affinity compared to free YefM. Operator interaction by YefM and YefM–YoeB is accompanied by structural transitions in the proteins. Paired 5′-TGTACA-3′ motifs are common in yefM-yoeB regulatory regions in diverse genomes suggesting that interaction of YefM–YoeB with these motifs is a conserved mechanism of operon autoregulation. Artificial perturbation of transcriptional autorepression could elicit inappropriate YoeB toxin production and induction of bacterial cell suicide, a potentially novel antibacterial strategy.

Structural and functional analysis of RNA and TAP binding to SF2/ASF

The serine/arginine‐rich (SR) protein splicing factor 2/alternative splicing factor (SF2/ASF) has a role in splicing, stability, export and translation of messenger RNA. Here, we present the structure of the RNA recognition motif (RRM) 2 from SF2/ASF, which has an RRM fold with a considerably extended loop 5 region, containing a two‐stranded β‐sheet. The loop 5 extension places the previously identified SR protein kinase 1 docking sequence largely within the RRM fold. We show that RRM2 binds to RNA in a new way, by using a tryptophan within a conserved SWQLKD motif that resides on helix α1, together with amino acids from strand β2 and a histidine on loop 5. The linker connecting RRM1 and RRM2 contains arginine residues, which provide a binding site for the mRNA export factor TAP, and when TAP binds to this region it displaces RNA bound to RRM2.

Binding of UNC-18 to the N-terminus of syntaxin is essential for neurotransmission in Caenorhabditis elegans

SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) are widely accepted to drive all intracellular membrane fusion events. SM (Sec1/Munc18-like) proteins bind to SNAREs and this interaction may underlie their ubiquitous requirement for efficient membrane fusion. SM proteins bind to SNAREs in at least three modes: (i) to a closed conformation of syntaxin; (ii) to the syntaxin N-terminus; and (iii) to the assembled SNARE complex. Munc18-1 exhibits all three binding modes and recent in vitro reconstitution assays suggest that its interaction with the syntaxin N-terminus is essential for neuronal SNARE complex binding and efficient membrane fusion. To investigate the physiological relevance of these binding modes, we studied the UNC-18/UNC-64 SM/SNARE pair, which is essential for neuronal exocytosis in Caenorhabditis elegans. Mutations in the N-terminus of UNC-64 strongly inhibited binding to UNC-18, as did mutations targeting closed conformation binding. Complementary mutations in UNC-18 designed to selectively impair binding to either closed syntaxin or its N-terminus produced a similarly strong inhibition of UNC-64 binding. Therefore high-affinity UNC18/UNC-64 interaction in vitro involves both binding modes. To determine the physiological relevance of each mode, unc-18-null mutant worms were transformed with wild-type or mutant unc-18 constructs. The UNC-18(R39C) construct, that is defective in closed syntaxin binding, fully rescued the locomotion defects of the unc-18 mutant. In contrast, the UNC-18(F113R) construct, that is defective in binding to the N-terminus of UNC-64, provided no rescue. These results suggest that binding of UNC-18 to closed syntaxin is dispensable for membrane fusion, whereas interaction with the syntaxin N-terminus is essential for neuronal exocytosis in vivo.

Cooperative Catalysis through Noncovalent Interactions

Noncovalent interactions, such as hydrogen bonding, electrostatic, p–p, CH–p, and hydrophobic forces, play an essential role in the action of nature s catalysts, enzymes. In the last decade these interactions have been successfully exploited in organocatalysis with small organic molecules. In contrast, such interactions have rarely been studied in the wellestablished area of organometallic catalysis, where electronic interactions through covalent bonding and steric effects imposed by bound ligands dictate the activity and selectivity of a metal catalyst. An interesting question is: What happens when an organocatalyst meets an organometallic catalyst? This unification has already created an exciting new space for both fields: cooperative catalysis, where reactants are activated simultaneously by both types of catalyst, thereby enabling reactivity and selectivity patterns inaccessible within each field alone. However, the mechanisms by which the two catalysts cooperatively effect the catalysis remain to be delineated. We recently found that combining an achiral iridium catalyst with a chiral phosphoric acid allows for highly enantioselective hydrogenation of imines (Scheme 1). To gain insight into the mechanism of this metal–organo cooperative catalysis, we studied the catalytic system with a range of techniques, including high pressure 2D-NMR spectroscopy, diffusion measurements, and NOEconstrained computation. Herein we report our findings. To evaluate the mechanism, a simplified achiral complex C was used, which leads to [C][A ] upon mixing, in situ or ex situ, with the chiral phosphoric acid HA through protonation at the amido nitrogen (Scheme 1). In the asymmetric hydrogenation of the model ketimine 1a, [C][A ] afforded 95% ee and full conversion. On the basis of related studies, the hydrogenation can be broadly explained by the catalytic cycle shown in Scheme 1, that is, [C][A ] activates H2 to give the hydride D and protonated 1a, which forms an ion pair with the phosphate affording [1a][A ]; hydride transfer furnishes the amine product 2a while regenerating [C][A ]. Questions pertinent to possible iridium–phosphate cooperation then arise: 1) How does the chiral phosphoric acid induce asymmetry in the hydrogenation? and 2) Does the enantioselectivity result from D being formed enantioselectively from [C][A ], from the phosphate salt [1a][A ], or from interactions involving all three components? We looked first at how the formation of hydride D and its transfer into the substrate are influenced by the chiral acid HA. The studies were carried out in CH2Cl2 or CD2Cl2 owing to the low solubility of the various metal complexes in toluene. The catalytic hydrogenation is feasible in both solvents, giving a 95% ee in toluene and 85 % ee in CH2Cl2 in the case of hydrogenation of 1a with C and HA under the conditions given in Scheme 1. The solution NMR studies show that the ionic complex [C][A ] is formed instantly on protonation of C (0.05 mmol) with one equivalent HA in CD2Cl2 (0.5 mL). Under H2 pressure (> 1 bar), proton transfer from a [C]–H2 dihydrogen intermediate (not observed) to 1a converts [C] into the hydride D and affords the salt [1a] [A ]. Formation of D took place instantly even at 78 8C, and it is observed during catalytic turnover, thus indicating that the hydrogenation is rate-limited by the hydride transfer step. Scheme 1. Hydrogenation of imine with achiral C and chiral acid HA (PMP = p-methoxyphenyl, Ar= 2,4,6-triisopropylphenyl, Ts = tosyl, Bn = benzyl).