Predicting Protein Structure With Probabilistic Models

Stultz, Collin M.; Nambudripad, Raman; Lathrop, Richard H.; White, James V.

doi:10.1016/s1569-2558(08)60483-x

Cited by 37 publications

(32 citation statements)

References 54 publications

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“…Examination of the hydropathy profile of the fourth extracellular domain of bovine rhodopsin using SYBYL6.6 demonstrated that the hydrophobicity of the sequence is markedly less in the region of the extracellular loop as compared with the adjoining helical regions. This was confirmed by protein sequence analysis (45)(46)(47). Hydropathy evaluation and protein sequence analysis were used to locate the fourth extracellular domain regions in both mouse GRPR and rat NMBR.…”

Section: Methodsmentioning

confidence: 84%

Molecular Basis for Selectivity of High Affinity Peptide Antagonists for the Gastrin-releasing Peptide Receptor

Tokita¹,

Katsuno²,

Hocart³

et al. 2001

Journal of Biological Chemistry

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]Bn(6 -14)), and a pseudopeptide analogue, JMV641 (D-Phe-Gln-Trp-Ala-Val-GlyHis-Leu(CHOH-CH 2 )-(CH 2 ) 2 -CH 3 ), were studied. Each had high affinity for the GRPR and >3,000-fold selectivity for GRPR over the closely related neuromedin B receptor (NMBR). To investigate the basis for this, we used a chimeric receptor approach to make both GRPR loss of affinity and NMBR gain of affinity chimeras and a site-directed mutagenesis approach. Chimeric or mutated receptors were transiently expressed in Balb/c 3T3. Only substitution of the fourth extracellular (EC) domain of the GRPR by the comparable NMBR domain markedly decreased the affinity for both antagonists. Substituting the fourth EC domain of NMBR into the GRPR resulted in a 300-fold gain in affinity for JMV594 and an 11-fold gain for JMV641. Each of the 11 amino acid differences between the GRPR and NMBR in this domain were exchanged. in GRPR by the three comparable NMBR amino acids caused a 500-fold decrease in affinity for both antagonists. Replacing the comparable three amino acids in NMBR by those from GRPR caused a gain in affinity for each antagonist. Receptor modeling showed that each of these three amino acids faced inward and was within 5 Å of the putative binding pocket. These results demonstrate that differences in the fourth EC domain of the mammalian Bn receptors are responsible for the selectivity of these two peptide antagonists. They demonstrate that Thr 297 , Phe 302 , and Ser 305 of the fourth EC domain of GRPR are the critical residues for determining GRPR selectivity and suggest that both receptor-ligand cation-interactions and hydrogen bonding are important for their high affinity interaction.The gastrin-releasing peptide (GRP) 1 receptor, which mediates the diverse actions of the mammalian bombesin (Bn)-related peptide (1, 2), GRP, has numerous high affinity peptide antagonists (3-5). This is in contrast to most other gastrointestinal (GI) hormone/neurotransmitter receptors for which no high affinity peptide antagonists exist (6). These GRP receptor antagonists are now widely used in both in vitro studies (5) and in vivo studies in animals (3, 7-11) and humans (12). Recent studies show that for many nonpeptide antagonists, differences in amino acids in the transmembrane domains between receptor subtypes are frequently particularly important for determining receptor subtype selectivity (13,14). A recent study (15) shows a similar result with the peptoid antagonist PD168368 for the neuromedin B receptor. However, with peptide antagonists of non-GI hormone/neurotransmitter receptors, interactions with transmembrane regions (16, 17) or extracellular domains (18) are important for high affinity interaction or receptor subtype selectivity. Which if any of these results apply to the different classes of GRPR peptide antagonists, at present, is unclear.GRP and neuromedin B (NMB), mammalian homologues of the amphibian tetradecapeptide bombesin, have structurally related carboxyl termini (19). These peptides mediate a spectrum of biological activi...

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Section: Methodsmentioning

confidence: 84%

Molecular Basis for Selectivity of High Affinity Peptide Antagonists for the Gastrin-releasing Peptide Receptor

Tokita¹,

Katsuno²,

Hocart³

et al. 2001

Journal of Biological Chemistry

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“…Both Trm8 and Trm82 are widely conserved proteins that have not previously been ascribed a function, as determined by a search of the Saccharomyces Genome Database (Cherry et al+, 2002), SwissProt (Gasteiger et al+, 2001), TrEMBL (Bairoch & Apweiler, 2000), and CDD (Marchler-Bauer et al+, 2002)+ A BLAST search (Altschul et al+, 1997) (Stultz et al+, 1997), which are often implicated in macromolecular interaction and regulatory functions (Neer et al+, 1994)+ Neither Trm8 nor Trm82 is significantly related to KgmB from S. tenebrarius, a rRNA m 7 G methyltransferase associated with aminoglycoside resistance (Beauclerk & Cundliffe, 1987;Holmes & Cundliffe, 1991) or to Abd1, which catalyzes m 7 G formation in mRNA cap structures (Mao et al+, 1995), other than the methyltransferase domain shared by Trm8, Abd1, and numerous other methyltransferases (Niewmierzycka & Clarke, 1999)+ Diploid strains carrying homozygous deletions of either TRM8 or TRM82 are viable (Winzeler et al+, 1999), and closer examination of growth reveals no measurable growth rate differences, compared to wild-type control strains, in rich media containing glucose at 30 8C+ Strains lacking either Trm8 or Trm82 yield extracts with no detectable tRNA m 7 G methyltransferase activity, and have severely reduced m 7 G-modified tRNA in vivo…”

Section: Database Analysis Of Trm8 and Trm82 Proteinsmentioning

confidence: 99%

“…Sequences were aligned using Mutalin (Corpet, 1988), and shadowed with Boxshade 3+21+ The WD40 motifs were predicted with the program Protein Sequence Analysis (PSA) http://bmerc-www+bu+edu/psa/request+htm (Stultz et al+, 1997)+…”

Section: Software Usedmentioning

confidence: 99%

Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA

Alexandrov

Martzen

Phizicky

2002

RNA

268

278

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ABSTRACT7-methylguanosine (m 7 G) modification of tRNA occurs widely in eukaryotes and bacteria, is nearly always found at position 46, and is one of the few modifications that confers a positive charge to the base. Screening of a Saccharomyces cerevisiae genomic library of purified GST-ORF fusion proteins reveals two previously uncharacterized proteins that copurify with m 7 G methyltransferase activity on pre-tRNA Phe . ORF YDL201w encodes Trm8, a protein that is highly conserved in prokaryotes and eukaryotes and that contains an S-adenosylmethionine binding domain. ORF YDR165w encodes Trm82, a less highly conserved protein containing putative WD40 repeats, which are often implicated in macromolecular interactions. Neither protein has significant sequence similarity to yeast Abd1, which catalyzes m 7 G modification of the 59 cap of mRNA, other than the methyltransferase motif shared by Trm8 and Abd1. Several lines of evidence indicate that both Trm8 and Trm82 proteins are required for tRNA m 7 G-methyltransferase activity: Extracts derived from strains lacking either gene have undetectable m 7 G methyltransferase activity, RNA from strains lacking either gene have much reduced m 7 G, and coexpression of both proteins is required to overproduce activity. Aniline cleavage mapping shows that Trm8/Trm82 proteins modify pre-tRNA Phe at G46, the site that is modified in vivo. Trm8 and Trm82 proteins form a complex, as affinity purification of Trm8 protein causes copurification of Trm82 protein in approximate equimolar yield. This functional two-protein family appears to be retained in eukaryotes, as expression of both corresponding human proteins, METTL1 and WDR4, is required for m 7 Gmethyltransferase activity.

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“…ary structure were obtained using software available on the protein sequence analysis server at the Biomolecular Engineering Research Center of Boston University (45).…”

Section: Structural Conservation Of Variable Borrelia Lipoproteinsmentioning

confidence: 99%

Structural Conservation of Neurotropism-associated VspA within the Variable Borrelia Vsp-OspC Lipoprotein Family

Zückert

Kerentseva

Lawson

et al. 2001

Journal of Biological Chemistry

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Vsp surface lipoproteins are serotype-defining antigens of relapsing fever spirochetes that undergo multiphasic antigenic variation to avoid the immune response. One of these proteins, VspA of Borrelia turicatae, is also associated with neurotropism in infected mice. Vsp proteins are highly polymorphic in sequence, which may relate to their specific antibody reactivities and host cell interactions. To determine whether sequence variations affect protein structure, we compared B. turicatae VspA with three related proteins: VspB of B. turicatae, Vsp26 of the relapsing fever agent Borrelia hermsii, and OspC of the Lyme disease spirochete Borrelia burgdorferi. Recombinant non-lipidated proteins were purified by affinity or ion exchange chromatography. Circular dichroism spectra revealed similar, highly ␣-helical secondary structures for all four proteins. In vitro assays demonstrated proteaseresistant, thermostable Vsp cores starting at a conserved serine at position 34 (Ser 34 ). All proteins aggregate as dimers in solution. In situ trypsin treatment and surface protein cross-linking showed that the native lipoproteins also form protease-resistant dimers. These findings indicate that Vsp proteins have a common compact fold and that their established functions are based on localized polymorphisms. Two forms of VspA crystals suitable for structure determination by x-ray diffraction methods have been obtained.

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Predicting Protein Structure With Probabilistic Models

Cited by 37 publications

References 54 publications

Molecular Basis for Selectivity of High Affinity Peptide Antagonists for the Gastrin-releasing Peptide Receptor

Molecular Basis for Selectivity of High Affinity Peptide Antagonists for the Gastrin-releasing Peptide Receptor

Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA

Structural Conservation of Neurotropism-associated VspA within the Variable Borrelia Vsp-OspC Lipoprotein Family

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