Structure of the transition state in the folding process of human procarboxypeptidase A2 activation domain

Villegas, Sandra; Martínez, José C.; Avilés, Francesc X.; Serrano, Luís

doi:10.1006/jmbi.1998.2158

Cited by 164 publications

(172 citation statements)

References 37 publications

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“…On the other hand, changes in the main inhibition loop of pro-CPB, which connects ␤2 and ␤3 chains at the globular core of the pro-segment, resulted in low expression levels. This is in agreement with other studies on the folding of isolated human pro-CPA2 activation domain (34), which demonstrated that mutations in such a region affect the folding capability of the pro-segment.…”

Section: Discussionsupporting

confidence: 93%

Mapping the Pro-region of Carboxypeptidase B by Protein Engineering

Ventura¹,

Villegas²,

Sterner³

et al. 1999

Journal of Biological Chemistry

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The proteolytic processing of pancreatic procarboxypeptidase B to a mature and functional enzyme is much faster than that of procarboxypeptidase A1. This different behavior has been proposed to depend on specific conformational features at the region that connects the globular domain of the pro-segment to the enzyme and at the contacting surfaces on both moieties. A cDNA coding for porcine procarboxypeptidase B was cloned, sequenced, and expressed at high yield (250 mg/liter) in the methylotrophic yeast Pichia pastoris. To test the previous hypothesis, different mutants of the pro-segment at the putative tryptic targets in its connecting region and at some of the residues contacting the active enzyme were obtained. Moreover, the complete connecting region was replaced by the homologous sequence in procarboxypeptidase A1. The detailed study of the tryptic processing of the mutants shows that limited proteolysis of procarboxypeptidase B is a very specific process, as Arg-95 is the only residue accessible to tryptic attack in the proenzyme. A fast destabilization of the connecting region after the first tryptic cut allows subsequent proteolytic processing and the expression of carboxypeptidase B activity. Although all pancreatic procarboxypeptidases have a preformed active site, only the A forms show intrinsic activity. Mutational substitution of Asp-41 in the globular activation domain, located at the interface with the enzyme moiety, as well as removal of the adjacent 3 10 helix allow the appearance of residual activity in the mutated procarboxypeptidase B, indicating that the interaction of both structural elements with the enzyme moiety prevents the binding of substrates and promotes enzyme inhibition. In addition, the poor heterologous expression of such mutants indicates that the mutated region is important for the folding of the whole proenzyme.

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Section: Discussionsupporting

confidence: 93%

Mapping the Pro-region of Carboxypeptidase B by Protein Engineering

Ventura¹,

Villegas²,

Sterner³

et al. 1999

Journal of Biological Chemistry

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“…So far, it has been confirmed that the predicted compact regions by ADMs for myoglobin and plant leghemoglobin [25], fatty acid binding proteins [25] and ferredoxin-like fold [30] proteins correspond well to the early folding regions detected by NMR studies [34,35], kinetic studies [36] and ϕ-value analyses [37][38][39][40].…”

Section: Analyses With Inter-residue Average Distance Statistics Conmentioning

confidence: 80%

Detection of Folding Sites of β-Trefoil Fold Proteins Based on Amino Acid Sequence Analyses and Structure-Based Sequence Alignment

Kirioka¹,

Aumpuchin²,

Kikuchi³

2017

J Proteomics Bioinform

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“…Clearly the choice of which hairpin is formed first is determined by the sequence (30,34). Other examples of homologous proteins in which the transition states consist of different parts of the protein are U1A, S6, and Ada2h (35,36).…”

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confidence: 99%

Probing the folding free energy landscape of the src-SH3 protein domain

Shea

Onuchic

Brooks

2002

Proc. Natl. Acad. Sci. U.S.A.

138

127

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The mechanism and thermodynamics of folding of the Src homology 3 (SH3) protein domain are characterized at an atomic level through molecular dynamics with importance sampling. This methodology enables the construction of the folding free energy landscape of the protein as a function of representative reaction coordinates. We observe that folding proceeds in a downhill manner under native conditions, with early compaction and structure formation in the hydrophobic sheet consisting of the three central ␤ strands of the protein. This state bears considerable resemblance to the experimentally determined transition state for folding. Folding proceeds further with the formation of the second hydrophobic sheet consisting of the terminal strands and the RT loop. The final stages of folding appear to involve the formation of the hydrophobic core through the expulsion of water molecules bridging the two hydrophobic sheets. This work sheds new light on the complementary roles of sequence and topology in governing the folding mechanism of small proteins and provides further support for the role of water in facilitating the late stages in folding.protein folding ͉ folding mechanism ͉ frustration ͉ topology

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Structure of the transition state in the folding process of human procarboxypeptidase A2 activation domain

Cited by 164 publications

References 37 publications

Mapping the Pro-region of Carboxypeptidase B by Protein Engineering

Mapping the Pro-region of Carboxypeptidase B by Protein Engineering

Detection of Folding Sites of β-Trefoil Fold Proteins Based on Amino Acid Sequence Analyses and Structure-Based Sequence Alignment

Probing the folding free energy landscape of the src-SH3 protein domain

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