Noroviruses are the major cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.7 Å resolution. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, which is based on the most rapidly cleaved recognition sequence in the 200 kDa polyprotein substrate, reacts covalently through its propenyl ethyl ester group (X) with the active site nucleophile, Cys 139. The structure permits, for the first time, the identification of substrate recognition and binding groups in a noroviral 3C protease and thus provides important new information for the development of antiviral prophylactics.

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Sarcolipin (SLN) is a small peptide found in the sarcoplasmic reticulum of skeletal muscle. It is predicted to contain a single hydrophobic transmembrane alpha-helix. Fluorescence emission spectra for the single Trp residue of SLN suggest that SLN incorporates fully into bilayers of dioleoylphosphatidylcholine, but only partially into bilayers of phosphatidylcholines with long (C(22) or C(24)) fatty acyl chains. The fluorescence of SLN is quenched in bilayers of dibromostearoylphosphatidylcholine, also consistent with incorporation into the lipid bilayer. SLN was reconstituted with the Ca(2+)-ATPase of skeletal-muscle sarcoplasmic reticulum. Even at a 50:1 molar ratio of SLN/ATPase, SLN had no significant effect on the rate of ATP hydrolysis by the ATPase or on the Ca(2+)-dependence of ATP hydrolysis. However, at a molar ratio of SLN/ATPase of 2:1 or higher the presence of SLN resulted in a marked decrease in the level of accumulation of Ca(2+) by reconstituted vesicles. The effect of SLN was structurally specific and did not result from a breakdown in the vesicular structure or from the formation of non-specific ion channels. Vesicles were impermeable to Ca(2+) in the absence of ATP in the external medium. The effects of SLN on accumulation of Ca(2+) can be simulated assuming that SLN increases the rate of slippage on the ATPase and the rate of passive leak of Ca(2+) mediated by the ATPase. It is suggested that the presence of SLN could be important in non-shivering thermogenesis, a process in which heat is generated by hydrolysis of ATP by skeletal-muscle sarcoplasmic reticulum.

We have developed a fluorescence quenching method using peptides containing 3,5-dibromotryrosine to measure oligomerization of model transmembrane alpha-helices in lipid bilayers. Peptides of the type Ac-LysLysGlyLeu(m)XLeu(n)LysLysAla-amide where X is tryptophan or 3,5-dibromotyrosine were found to form heterodimers in bilayers of phosphatidylcholine in the liquid-crystalline phase. The free energy of dimer formation changed little with increasing number of Leu residues from 16 to 22 but increased with increasing phospholipid fatty acyl chain length, with a slope of about 0.5 kJ mol(-1) per fatty acyl chain carbon. Peptides were excluded from lipid in the gel phase, resulting in increased levels of oligomerization. Addition of cholesterol to form the liquid-ordered state led to increased dimerization but without phase separation. The presence of phosphatidylethanolamine had little effect on dimerization.

We have studied the effects of aromatic residues at the ends of peptides of the type Ac-KKGL(n)()WL(m)()KKA-amide on their interactions with lipid bilayers as a function of lipid fatty acyl chain length, physical phase, and charge. Peptide Ac-KKGFL(6)WL(8)FKKA-amide (F(2)L(14)) incorporated into bilayers of phosphatidylcholines containing monounsaturated fatty acyl chains of lengths C14-C24 at a peptide:lipid molar ratio of 1:100 in contrast to Ac-KKGL(7)WL(9)KKA-amide (L(16)) which did not incorporate at all into dierucoylphosphatidylcholine [di(C24:1)PC]; Ac-KKGYL(6)WL(8)YKKA-amide (Y(2)L(14)) incorporated partly into di(C24:1)PC. Lipid-binding constants relative to that for dioleoylphosphatidylcholine (C18:1)PC were obtained using a fluorescence quenching method. For Y(2)L(14) and F(2)L(14), relative lipid-binding constants increased with increasing fatty acyl chain length from C14 to C24; strongest binding did not occur at the point where the hydrophobic length of the peptide equalled the hydrophobic thickness of the bilayer. For Ac-KKGYL(9)WL(11)YKKA-amide (Y(2)L(20)), increasing chain length from C18 to C24 had little effect on relative binding constants. Anionic phospholipids bound more strongly than zwitterionic phospholipids to Y(2)L(14) and Y(2)L(20) but effects of charge were relatively small. In two phase (gel and liquid crystalline) mixtures, all the peptides partitioned more strongly into liquid crystalline than gel phase; effects were independent of the structure of the peptide or of the lipid (dipalmitoylphosphatidylcholine or bovine brain sphingomyelin). Addition of cholesterol had little effect on incorporation of the peptides into lipid bilayers. It is concluded that the presence of aromatic residues at the ends of transmembrane alpha-helices effectively buffers them against changes in bilayer thickness caused either by an increase in the chain length of the phospholipid or by the presence of cholesterol.

Sarcolipin (SLN) is a small peptide found in the sarcoplasmic reticulum of skeletal muscle. It is predicted to contain a single hydrophobic transmembrane α-helix. Fluorescence emission spectra for the single Trp residue of SLN suggest that SLN incorporates fully into bilayers of dioleoylphosphatidylcholine, but only partially into bilayers of phosphatidylcholines with long (C22 or C24) fatty acyl chains. The fluorescence of SLN is quenched in bilayers of dibromostearoylphosphatidylcholine, also consistent with incorporation into the lipid bilayer. SLN was reconstituted with the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum. Even at a 50:1 molar ratio of SLN/ATPase, SLN had no significant effect on the rate of ATP hydrolysis by the ATPase or on the Ca2+-dependence of ATP hydrolysis. However, at a molar ratio of SLN/ATPase of 2:1 or higher the presence of SLN resulted in a marked decrease in the level of accumulation of Ca2+ by reconstituted vesicles. The effect of SLN was structurally specific and did not result from a breakdown in the vesicular structure or from the formation of non-specific ion channels. Vesicles were impermeable to Ca2+ in the absence of ATP in the external medium. The effects of SLN on accumulation of Ca2+ can be simulated assuming that SLN increases the rate of slippage on the ATPase and the rate of passive leak of Ca2+ mediated by the ATPase. It is suggested that the presence of SLN could be important in non-shivering thermogenesis, a process in which heat is generated by hydrolysis of ATP by skeletal-muscle sarcoplasmic reticulum.

We report experiments to investigate the role of the physiologically relevant protein tyrosine kinase Lck in the ordered phosphorylation of the T-cell receptor f chain. Six synthetic peptides were designed based on the sequences of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the f chain. Preliminary 1 H-NMR studies of recombinant f chain suggested that it is essentially unstructured and therefore that peptide mimics would serve as useful models for investigating individual ITAM tyrosines. Phosphorylation kinetics were determined for each tyrosine by assaying the transfer of 32 P by recombinant Lck on to each of the peptides. The rates of phosphorylation were found to depend on the location of the tyrosine, leading to the proposal that Lck phosphorylates the six f chain ITAM tyrosines in the order 1N (first) > 3N > 3C > 2N > 1C > 2C (last) as a result of differences in the amino-acid sequence surrounding each tyrosine. This proposal was then tested on cytosolic, recombinant T-cell receptor f chain. After in vitro phosphorylation by Lck, the partially phosphorylated f chain was digested with trypsin. Separation and identification of the f chain fragments using LC-MS showed, as predicted by the peptide phosphorylation studies, that tyrosine 1N is indeed the first to be phosphorylated by Lck. We conclude that differences in the amino-acid context of the six f chain ITAM tyrosines affect the efficiency of their phosphorylation by the kinase Lck, which probably contributes to the distinct patterns of phosphorylation observed in vivo.

HighlightsSarcolipin is insufficient to affect thermogenic activity of SERCA in mouse muscle.The ratio of SLN to SERCA in total limb skeletal muscle is <0.0015 mol/mol.Knocking out this SLN in mice would have a small effect on SERCA function.Overexpressing SLN in transgenic mice only resulted in 0.037 mol SLN/mol SERCA.SLN+/+ mice showed no evidence of an increase in thermogenesis.