Phospholipase A2 Engineering. The Roles of Disulfide Bonds in Structure, Conformational Stability, and Catalytic Function

Zhu, Hongxin; Dupureur, Cynthia M.; Zhang, Xiaoyan; Tsai, Ming‐Daw

doi:10.1021/bi00046a040

Cited by 64 publications

(63 citation statements)

References 36 publications

(62 reference statements)

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“…The loss of entropy in the unfolded state is not, however, the only effect of disulfide bonds on conformational stability. A disulfide bond can enhance (39 -41) or diminish (42)(43)(44)(45) interactions in the folded state that contribute to conformational stability. Thus, predicting the precise contribution of a particular native or nonnative disulfide bond to conformational stability is difficult.…”

Section: Discussionmentioning

confidence: 99%

Conformational Stability Is a Determinant of Ribonuclease A Cytotoxicity

Klink¹,

Raines²

2000

Journal of Biological Chemistry

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Onconase™, a homolog of bovine pancreatic ribonuclease A (RNase A) with high conformational stability, is cytotoxic and has efficacy as a cancer chemotherapeutic agent. Unlike wild-type RNase A, the G88R variant is toxic to cancer cells. Here, variants in which disulfide bonds were removed from or added to G88R RNase A were used to probe the relationship between conformational stability and cytotoxicity in a methodical manner. The conformational stability of the C40A/G88R/C95A and C65A/C72A/G88R variants is less than that of G88R RNase A. In contrast, a new disulfide bond that links the N and C termini (residues 4 and 118) increases the conformational stability of G88R RNase A and C65A/C72A/ G88R RNase A. These changes have little effect on the ribonucleolytic activity of the enzyme or on its ability to evade the cytosolic ribonuclease inhibitor protein. The changes do, however, have a substantial effect on toxicity toward human erythroleukemia cells. Specifically, conformational stability correlates directly with cytotoxicity as well as with resistance to proteolysis. These data indicate that conformational stability is a key determinant of RNase A cytotoxicity and suggest that cytotoxicity relies on avoiding proteolysis. This finding suggests a means to produce new cancer chemotherapeutic agents based on mammalian ribonucleases. The free energy difference between the native and unfolded states of a protein is small-typically 5-15 kcal/mol (1, 2). In their native state, many proteins are less susceptible to proteolytic degradation than when unfolded (3). In the unfolded state, the steric protection of peptide bonds provided by the compact native state is lost (4). For example, bovine pancreatic ribonuclease A (RNase A) 1 (EC 3.1.27.5 (5, 6)) is degraded more readily by proteases in the presence of denaturants or elevated temperatures (7-10) and less readily when glycosylated (11).The rates of intracellular protein turnover vary by 10 3 -fold (12). Apparently, some proteins are better able to thwart the proteolytic machinery (13,14). These proteins remain intact and retain activity longer within the cell. A correlation between the conformational stability of a protein and its catabolism was reported over 20 years ago (15). Since then, studies using unrelated proteins have either supported (16) or contradicted (14, 17) this correlation. Drawing conclusions from these studies is problematic because the proteins were divergent in characteristics that have been implicated in metabolic turnover. In contrast, variants of a single protein, the N-terminal domain of the repressor protein from bacteriophage , have been used to demonstrate a definite link between conformational stability and metabolic turnover (18). More recently, the conformational stability of bovine pancreatic trypsin inhibitor variants was shown to correlate with the yield of intact protein produced in a heterologous system (19).RNase A homologs elicit diverse biological activities, including specific toxicity to cancer cells (20,21). Onconase™, which ...

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Section: Discussionmentioning

confidence: 99%

Conformational Stability Is a Determinant of Ribonuclease A Cytotoxicity

Klink¹,

Raines²

2000

Journal of Biological Chemistry

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“…Additionally, no AR protein or an AR repeat domain has been found to contain a disulfide bond, a structural element that could stabilize a protein but may not always be advantageous (31). Hence, one might think that AR proteins differ from globular proteins in folding and stability and each AR in a protein could unfold and refold individually.…”

Section: Folding and Stabilitymentioning

confidence: 99%

Ankyrin Repeat: A Unique Motif Mediating Protein−Protein Interactions

2006

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Ankyrin repeat, one of the most widely existing protein motifs in nature, consists of 30−34 amino acid residues and exclusively functions to mediate protein−protein interactions, some of which are directly involved in the development of human cancer and other diseases. Each ankyrin repeat exhibits a helix−turn−helix conformation, and strings of such tandem repeats are packed in a nearly linear array to form helix−turn−helix bundles with relatively flexible loops. The global structure of an ankyrin repeat protein is mainly stabilized by intra- and inter-repeat hydrophobic and hydrogen bonding interactions. The repetitive and elongated nature of ankyrin repeat proteins provides the molecular bases of the unique characteristics of ankyrin repeat proteins in protein stability, folding and unfolding, and binding specificity. Recent studies have demonstrated that ankyrin repeat proteins do not recognize specific sequences, and interacting residues are discontinuously dispersed into the whole molecules of both the ankyrin repeat protein and its partner. In addition, the availability of thousands of ankyrin repeat sequences has made it feasible to use rational design to modify the specificity and stability of physiologically important ankyrin repeat proteins and even to generate ankyrin repeat proteins with novel functions through combinatorial chemistry approaches.

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“…The conformational rigidity of eukaryotic PLA 2 s is partially accomplished by a high content of disulfide bonds (32). However, the S. violaceoruber PLA 2 has only two disulfide bonds.…”

Section: Quality Of the Electron Density Map And The Current Model-asmentioning

confidence: 99%

The Crystal Structure of Prokaryotic Phospholipase A2

Matoba¹,

Katsube²,

Sugiyama³

2002

Journal of Biological Chemistry

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In this study, the x-ray crystal structures of the calcium-free and calcium-bound forms of phospholipase A 2 (PLA 2 ), produced extracellularly by Streptomyces violaceoruber, were determined by using the multiple isomorphous replacement and molecular replacement methods, respectively. The former and latter structures were refined to an R-factor of 18.8% at a 1.4-Å resolution and an R-factor of 15.0% at a 1.6-Å resolution, respectively. The overall structure of the prokaryotic PLA 2 exhibits a novel folding topology that demonstrates that it is completely distinct from those of eukaryotic PLA 2 s, which have been already determined by x-ray and NMR analyses. Furthermore, the coordination geometry of the calcium(II) ion apparently deviated from that of eukaryotic PLA 2 s. Regardless of the evolutionary divergence, the catalytic mechanism including the calcium(II) ion on secreted PLA 2 seems to be conserved between prokaryotic and eukaryotic cells. Demonstrating that the overall structure determined by x-ray analysis is almost the same as that determined by NMR analysis is useful to discuss the catalytic mechanism at the molecular level of the bacterial PLA 2 .

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Phospholipase A2 Engineering. The Roles of Disulfide Bonds in Structure, Conformational Stability, and Catalytic Function

Cited by 64 publications

References 36 publications

Conformational Stability Is a Determinant of Ribonuclease A Cytotoxicity

Conformational Stability Is a Determinant of Ribonuclease A Cytotoxicity

Ankyrin Repeat: A Unique Motif Mediating Protein−Protein Interactions

The Crystal Structure of Prokaryotic Phospholipase A2

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