Simon A. Sherman

The role of the SEA (sea urchin sperm protein, enterokinase and agrin) module in cleavage of membrane-tethered mucins

The membrane‐tethered mucins are cell surface‐associated dimeric or multimeric molecules with extracellular, transmembrane and cytoplasmic portions, that arise from cleavage of the primary polypeptide chain. Following the first cleavage, which may be cotranslational, the subunits remain closely associated through undefined noncovalent interactions. These mucins all share a common structural motif, the SEA module that is found in many other membrane‐associated proteins that are released from the cell surface and has been implicated in both the cleavage events and association of the subunits. Here we examine the SEA modules of three membrane‐tethered mucins, MUC1, MUC3 and MUC12, which have significant sequence homology within the SEA domain. We previously identified the primary cleavage site within the MUC1 SEA domain as FRPG/SVVV a sequence that is highly conserved in MUC3 and MUC12. We now show by site‐directed mutagenesis that the F, G and S residues are important for the efficiency of the cleavage reaction but not indispensable and that amino acids outside this motif are probably important. These data are consistent with a new model of the MUC1 SEA domain that is based on the solution structure of the MUC16 SEA module, derived by NMR spectroscopy. Further, we demonstrate that cleavage of human MUC3 and MUC12 occurs within the SEA domain. However, the SEA domains of MUC1, MUC3 and MUC12 are not interchangeable, suggesting that either these modules alone are insufficient to mediate efficient cleavage or that the 3D structure of the hybrid molecules does not adequately recreate an accessible cleavage site.

Identification of O-glycosylated decapeptides within the MUC1 repeat domain as potential MHC class I (A2) binding epitopes

The MUC1 glycoprotein is considered a tumor antigen due to its over expression and aberrant glycosylation in cancer tissues. The latter results in appearance of new antigenic tumor specific glycopeptides not found on normal glycoforms of the mucin. MUC1 glycopeptides can be presented by APCs on MHC class II molecules to activate glycopeptide specific helper T-cells. No study has yet reported presentation of MUC1 glycopeptides on MHC class I molecules as stimulators of cytotoxic T-cells. In this study we show that human immunoproteasomes and cathepsin-L can generate octa to undecameric glycopeptides from the MUC1 repeat domain in vitro. We identified glycosylated fragments of which the decameric glycopeptide SAP10 [SAPDT(GalNAc)RPAPG] containing a single sugar binds with comparable strength to the MHC class I allele HLA A*0201 as predicted high-score binding epitopes of the tandem repeat. The same sequence glycosylated with the disaccharide Gal-GalNAc does not bind. The glycan on SAP10 is predicted by molecular modeling to either protrude out or point into the MHC groove. SAPDTRPAPG peptide and the respective glycopeptide stimulated cytotoxic T-cells in vitro. Our findings suggest that MUC1 tandem repeat glycopeptides are capable of activating both helper and cytotoxic T-cells and thus represent good candidates for further development as vaccines.

Differential activities of decapeptide agonists of human C5a: the conformational effects of backbone N-methylation

Analogues of the potent, conformationally biased, decapeptide agonist of human C5a anaphylatoxin, C5a(65-74)Y65,F67,P69,P71,D-Ala73 (YSFKPMPLaR, peptide 54), were synthesized with methyl groups occupying specific amide nitrogen atoms along the peptide backbone. This N-methylation induced crucial extended backbone conformations in a manner similar to the two Pro residues, but without eliminating the contributions made by the side-chain of the residue for which Pro was substituted. The presence of backbone N-methyl groups on peptide 54 analogues had pronounced detrimental effects on the ability to bind and activate C5aRs expressed on human PMNs, but not on the ability to contract smooth muscle of human umbilical artery. Several N-methylated analogues of peptide 54 (peptides 56, 67, 124, 125, and 137) were significantly more selective for smooth muscle contraction, which is mediated by tissue resident macrophages, than for enzyme release from PMNs. Indeed, peptide 67, YSFKDMP(MeL)aR was almost 3000-fold more selective for smooth muscle contraction than for PMN enzyme release. Consistent with these differential activities was the observation that peptide 67 expressed a significantly greater binding affinity to C5aRs expressed on rat macrophages than on rat PMNs. This differential activity was also observed in vivo in the rat where peptide 67 induced a hypotensive response similar to peptide 54 and rhuC5a, but without accompanying neutropenia.

Comparison of Protein Structures in Solution Using Local Conformations Derived from NMR Data: Application to Cytochrome c

Structural comparisons of proteins in solution are often required to examine structure-functional relationships, study structural effects of mutations or distinguish between various forms of the same molecule under different conditions. A nuclear magnetic resonance (NMR) based probabilistic strategy is presented and used to study the structural differences between the two redox states of cytochrome c in solution. A probabilistic approach is employed to calculate the main chain conformations of horse ferro- and ferricytochrome c in solution, based on the published sequential d connectivity data. Conformational differences between the two oxidation states of horse cytochrome c in solution are found to be statistically significant. The largest changes in conformation are at residues Lys27, Thr28, Leu32, Gln42, Thr47, Tyr48, Thr49, Glu69, Lys72, Met80, Phe82, Ile85 and Lys86, all of which are close to the heme (within 14 A of the heme iron in the high resolution Xray structure of tuna cytochrome c). We suggest that these conformational changes may modulate local dipole moments and hence influence the interactions of cytochrome c with its physiological redox partners during the electron transfer process. The oxidation state dependent conformational differences are found to be much greater in solution than in the crystalline state, and the solution and crystal structures differ significantly in regions close to the heme. These results suggest that the highly charged nature of cytochrome c makes this protein particularly sensitive to the ionic strength of its environment and leads to differences between crystal and solution structures in the same oxidation state. In such cases, crystal structures must be used with caution for modeling molecular interactions in vivo. More generally, this analysis indicates that the determination of accurate local conformations based on nmr data can provide useful information about structure-functional aspects of proteins in solution.

A linear 23-residue peptide reveals a propensity to form an unusual native-like conformation.

To gain insight into the earliest events of protein folding, a 23-residue peptide with a sequence corresponding to the 38-60 fragment of the beta-subunit of human chorionic gonadotropin (hCG beta) was studied by NMR. In aqueous solution the majority of the peptide residues adopted an extended polyproline II (PII) conformation similar to those in mature, fully folded hCG beta. The finding that the isolated protein fragment may acquire native-like structural motifs, even without alpha-helices or beta-structures, extends the possibility of using free peptides as model systems to better understand the protein folding mechanisms. It was shown that the PII-rich structural motif can be determined efficiently by NMR spectroscopy. The observation that in the absence of extensive medium- and long-range interactions the majority of amino acid residues may adopt the PII conformation suggests that the PII-rich structural motifs may play an important role in early events of protein folding.

β-Hairpin stabilization in a 28-residue peptide derived from the β-subunit sequence of human chorionic gonadotropin hormone

The beta-subunit of the human chorionic gonadotropin (hCG) hormone, which is believed to be related to certain types of cancer, contains three hairpin-like fragments. To investigate the role of beta-hairpin formation in the early stages of the hCGbeta folding, a 28-residue peptide with the sequence RDVRFESIRLPGSPRGVNPVVSYAVALS, corresponding to the H3-beta hairpin fragment (residues 60-87) of the hCGbeta subunit, was studied under various conditions using three optical spectroscopic methods: Fourier transform ir spectroscopy, electronic CD, and vibrational CD. Environmental conditions are critical factors for formation of secondary structure in this peptide. TFE : H(2)O mixed solvents induced helical formation. Formation of beta-structure in this peptide, which may be related to the native beta-hairpin formation in the intact hormone, was found to be induced only under conditions such as high concentration, high temperature, and the presence of nonmicellar sodium dodecyl sulfate concentrations. These findings support a protein folding mechanism for the hCGbeta subunit in which an initial hydrophobic collapse, which increases intermolecular interactions in hCGbeta, is needed to induce the H3-beta hairpin formation.

Improvement in accuracy of protein local structure determination from NMR data

Abstract A method for determining the most probable conformations of amino acid residues from semiquantitatively estimated nuclear Overhauser effects (NOEs) and coupling constants was developed and coded in the FiSiNOE-2 program. This program is a new version of the FiSiNOE program, utilizing NMR data with complementary knowledge-based information on protein structures. In FiSiNOE-2 this information is conformational clusters of the dihedral angles (φ, ψ, gc 1 ) derived from the Protein Data Bank. The FiSiNOE-2 method determines mathematical expectations and standard deviations for the angles φ, ψ, and χ 1 , and provides direct determination of the local structure of proteins from NMR data before building and refining their spatial structure. The results of the FiSiNOE-2 program in combination with the results of the habas program may be used to provide stereospecific assignments of a pair of β-methylene protons and to determine precisely allowed ranges of the φ, ψ, and χ 1 dihedral angles consistent with a given set of NMR data. To do this, a new procedure, combine , was developed. Computational experiments with the NMR data simulated from X-ray coordinates of the BPTI showed that use of the combine procedure, in comparison with results obtained when habas was used alone, increases by more than 30% the number of correct assignments for βCH 2 groups and reduces the total lengths of the combined angular intervals for φ, ψ, and χ 1 angles to 1.9, 2.4, and 1.8 times, respectively. In contrast to the redundant dihedral angle constraints (REDAC) strategy, that derives REDAC from preliminary calculations of the complete structure, the combine procedure reduces the length of the angular intervals before using the variable target function algorithm to determine spatial structures of proteins. This feature of the combine strategy may be especially beneficial in the cases when there is lack of long-range NOEs.

A priori and a posteriori mixture distributions for using databases in protein structure determination

Abstract The twists and turns of protein molecules correspond to the rotational angles of the main and side chains in their constituent amino acid residues. Owing to stereochemical restrictions the joint distribution of these angles falls into several well-separated clusters. Consequently, we fit a statistical finite mixture model to data from the Brookhaven Protein Data Bank (PDB), obtaining a new classification of conformational states of amino acids. The results of this database modeling are important for knowledge-based approaches to protein structure determination and analysis and can be used in conjunction with experimental data in the determination of protein spatial structure. As part of this process, the mixture distributions we have fit can be used as a priori distributions. A posteriori distributions corresponding to these a priori mixture distributions are illustrated.