Sequence analysis and crystal structure of a glycosylated protease from Euphorbia resinifera latex for its proteolytic activity aspect

Using polyclonal antibodies raised against human pancreatic procarboxypeptidases, a full-length cDNA coding for an A-type proenzyme was isolated from a lambda gt11 human pancreatic library. This cDNA contains standard 3′ and 5′ flanking regions, a poly(A)+ tail and a central region of 1260 nucleotides coding for a protein of 419 amino acids. On the basis of sequence comparisons, the human protein was classified as a procarboxypeptidase A1 which is very similar to the previously described A1 forms from rat and bovine pancreatic glands. The presence of the amino acid sequences assumed to be of importance for the zymogen inhibition by its activation segment, primarily on the basis of the recently reported crystal structure of the B form, further supports the proposed classification.

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Chemical modification of GSH transferase P1-1 confirms the presence of Arg-13, Lys-44 and one carboxylate group in the GSH-binding domain of the active site

Biochemical Journal ◽

10.1042/bj2930357 ◽

1993 ◽

Vol 293 (2) ◽

pp. 357-362 ◽

Cited By ~ 22

Author(s):

C Xia ◽

D J Meyer ◽

H Chen ◽

P Reinemer ◽

R Huber ◽

...

Keyword(s):

Crystal Structure ◽

Sequence Analysis ◽

Active Site ◽

Sequence Similarity ◽

Lysine Residue ◽

Carboxylate Group ◽

Terminal Sequence ◽

Crystallographic Study ◽

Binding Determinants ◽

Subunit B

GSH transferase P1-1 (GSTP1-1) was modified with group-specific reagents. Kinetic experiments demonstrated that inactivation of GSTP1-1 occurred upon reaction of one arginine residue per subunit with diacetyl, one lysine residue per subunit with 2,4,6-trinitrobenzene sulphonate, or one carboxylate group per subunit with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. All three inactivation reactions were inhibited by compounds known to bind at the GSH site of the enzyme but were unaffected by the electrophile 1-chloro-2,4-dinitrobenzene. N-terminal sequence analysis showed that Arg-13 was modified by diacetyl and that this modification was inhibited by GSH. Arg-11 was not modified. The lysine residue modified by 2,4,6-trinitrobenzene sulphonate and protected by S-octylglutathione was identified as Lys-44 by sequencing of tryptic peptides. The findings are in agreement with the involvement of Arg-13 and Lys-44 in binding of GSH, as determined from the crystal structure [Reinemer, Dirr, Ladenstein, Huber, Lo Bello, Frederici and Parker (1992) J. Mol. Biol. 227, 214-226]. The present data also implicate a single carboxylate in GSH binding, consistent with the involvement of Asp-98 of subunit B determined from the crystallographic study. The GSH-binding determinants of GSTP1-1 are compared using sequence similarity with those of GSTs of Alpha, Mu and Theta classes.

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Comparison of the transmembrane helices of bovine rhodopsin in the crystal structure and the C α template based on cryo-electron microscopy maps and sequence analysis of the G protein-coupled receptors

Molecular Simulation ◽

10.1080/0892702021000002520 ◽

2002 ◽

Vol 28 (8-9) ◽

pp. 845-851 ◽

Cited By ~ 2

Author(s):

Siavoush Dastmalchi ◽

Felix J. Kobus ◽

Tiina P. Iismaa ◽

Michael B. Morris ◽

W. Bret Church

Keyword(s):

Crystal Structure ◽

Electron Microscopy ◽

Sequence Analysis ◽

G Protein ◽

G Protein Coupled Receptors ◽

Transmembrane Helices ◽

Cryo Electron Microscopy ◽

Bovine Rhodopsin ◽

G Protein Coupled

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Actin mutations that show suppression with fimbrin mutations identify a likely fimbrin-binding site on actin.

The Journal of Cell Biology ◽

10.1083/jcb.126.2.413 ◽

1994 ◽

Vol 126 (2) ◽

pp. 413-422 ◽

Cited By ~ 37

Author(s):

J E Honts ◽

T S Sandrock ◽

S M Brower ◽

J L O'Dell ◽

A E Adams

Keyword(s):

Crystal Structure ◽

Sequence Analysis ◽

Binding Site ◽

Actin Filaments ◽

Biochemical Analysis ◽

Actin Binding ◽

Cross Linking ◽

Temperature Sensitive ◽

Small Domain

Actin interacts with a large number of different proteins that modulate its assembly and mediate its functions. One such protein is the yeast actin-binding protein Sac6p, which is homologous to vertebrate fimbrin (Adams, A. E. M., D. Botstein, and D. G. Drubin. 1991. Nature (Lond.). 354:404-408.). Sac6p was originally identified both genetically (Adams, A. E. M., and D. Botstein. 1989. Genetics. 121:675-683.) by dominant, reciprocal suppression of a temperature-sensitive yeast actin mutation (act1-1), as well as biochemically (Drubin, D. G., K. G. Miller, and D. Botstein. 1988. J. Cell Biol. 107: 2551-2561.). To identify the region on actin that interacts with Sac6p, we have analyzed eight different act1 mutations that show suppression with sac6 mutant alleles, and have asked whether (a) these mutations occur in a small defined region on the crystal structure of actin; and (b) the mutant actins are defective in their interaction with Sac6p in vitro. Sequence analysis indicates that all of these mutations change residues that cluster in the small domain of the actin crystal structure, suggesting that this region is an important part of the Sac6p-binding domain. Biochemical analysis reveals defects in the ability of several of the mutant actins to bind Sac6p, and a reduction in Sac6p-induced cross-linking of mutant actin filaments. Together, these observations identify a likely site of interaction of fimbrin on actin.

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X-ray crystal structure of CMS1MS2: a high proteolytic activity cysteine proteinase from Carica candamarcensis

Amino Acids ◽

10.1007/s00726-012-1318-7 ◽

2012 ◽

Vol 43 (6) ◽

pp. 2381-2391 ◽

Cited By ~ 5

Author(s):

Marco T. R. Gomes ◽

Raphael D. Teixeira ◽

Míriam T. P. Lopes ◽

Ronaldo A. P. Nagem ◽

Carlos E. Salas

Keyword(s):

Crystal Structure ◽

Proteolytic Activity ◽

Cysteine Proteinase ◽

X Ray ◽

High Proteolytic Activity

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Structural evidence for a latch mechanism regulating access to the active site of SufS-family cysteine desulfurases

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320000790 ◽

2020 ◽

Vol 76 (3) ◽

pp. 291-301 ◽

Cited By ~ 2

Author(s):

Jack A. Dunkle ◽

Michael R. Bruno ◽

Patrick A. Frantom

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Sequence Analysis ◽

Active Site ◽

Resting State ◽

Sulfur Source ◽

Structural Elements ◽

Cysteine Desulfurase ◽

X Ray

Cysteine serves as the sulfur source for the biosynthesis of Fe–S clusters and thio-cofactors, molecules that are required for core metabolic processes in all organisms. Therefore, cysteine desulfurases, which mobilize sulfur for its incorporation into thio-cofactors by cleaving the Cα—S bond of cysteine, are ubiquitous in nature. SufS, a type 2 cysteine desulfurase that is present in plants and microorganisms, mobilizes sulfur from cysteine to the transpersulfurase SufE to initiate Fe–S biosynthesis. Here, a 1.5 Å resolution X-ray crystal structure of the Escherichia coli SufS homodimer is reported which adopts a state in which the two monomers are rotated relative to their resting state, displacing a β-hairpin from its typical position blocking transpersulfurase access to the SufS active site. A global structure and sequence analysis of SufS family members indicates that the active-site β-hairpin is likely to require adjacent structural elements to function as a β-latch regulating access to the SufS active site.

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