Comparison of the structural and functional properties of RNase A and BS‐RNase: A stepwise mutagenesis approach

Ercole, Carmine; Colamarino, Rosa Angela; Pizzo, Elio; Fogolari, Federico; Spadaccini, Roberta; Picone, Delia

doi:10.1002/bip.21176

Cited by 26 publications

(45 citation statements)

References 33 publications

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“…The insertion of two Cys residues, Pro19 and Leu28, is not sufficient to induce the RNase A dimeric variants to adopt the quaternary structure of NCD-BS. In the opened quaternary structure of these mutants, one subunit can be easily modeled into RI horseshoe cavity without steric interference produced by the partner chain (see Supporting Information Figure 1), as previously shown for other opened dimers 32 and for the more compact ND-RNase A molecule. 31 The analysis of the conformational preferences of the hinge peptide clearly suggests a strong synergic effect of the residues in the 16 and 19 position of the hinge peptide sequence.…”

Section: Discussionmentioning

confidence: 92%

“…The preparation and characterization of the GNPSCC are reported in an accompanying article. 32 GNPSCC also folds in the swapped and unswapped forms. The noncovalent dimers (NCD-LCC, NCD-PLCC, and NCD-GNPSCC) have been obtained from the equilibrium mixture (MxM and M5M) through selective reduction of interchain disulfide bridges, followed by alkylation of free thiol groups and by separation of monomer and dimer by using gel permeation chromatography.…”

Section: Recombinant Dna Methodologies and Protein Sample Preparationmentioning

confidence: 95%

“…In particular, three different mutants were prepared: LCC (Q28?L, K31?C, S32?C), PLCC (LCC with the additional substitution A19?P), and GNPSCC, which embodies the whole sequence of the seminal enzyme hinge peptide plus the two cysteines (S16?G, T17?N, A19?P, A20?S, K31?C, S32?C). 32 These mutants exist in the swapped and unswapped forms that, due to the intersubunit disulfide bridges, must adopt quaternary structures that are very similar to each other and very similar to those of the corresponding dimers of the seminal enzyme. 26 However, with respect to the latter protein, the three RNase A mutants are much less potent as cytotoxic agents.…”

Section: Introductionmentioning

confidence: 99%

“…26 However, with respect to the latter protein, the three RNase A mutants are much less potent as cytotoxic agents. 32 As the antitumoral properties of the seminal ribonuclease have been attributed to the noncovalent dimeric form, which is eventually produced in the cellular reducing environment, we have solved, for the three mutants, the crystal structures of the noncovalent dimeric form (NCD), which is critically dependent on the properties of the hinge loop. The structure of NCD-LCC has been determined in the presence of the substrate analogue 2 0 -de-…”

Section: Introductionmentioning

confidence: 99%

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Toward an antitumor form of bovine pancreatic ribonuclease: The crystal structure of three noncovalent dimeric mutants

et al. 2009

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The cytotoxic action of bovine seminal ribonuclease (BS-RNase) depends on its noncovalent swapped dimeric form (NCD-BS), which presents a compact structure that allows the molecule to escape ribonuclease inhibitor (RI). A key role in the acquisition of this structure has been attributed to the concomitant presence of a proline in position 19 and a leucine in position 28. The introduction of Leu28, Cys31, and Cys32 and, in addition, of Pro19 in the sequence of bovine pancreatic ribonuclease (RNase A) has produced two dimeric variants LCC and PLCC, which do exhibit a cytotoxic activity, though at a much lower level than BS-RNase. The crystal structure analysis of the noncovalent swapped form (NCD) of LCC and PLCC, complexed with the substrate analogue 2 '-deoxycytidylyl(3 ',5 ')-2 '-deoxyguanosine, has revealed that, differently from NCD-BS, the dimers adopt an opened quaternary structure, with the two Leu residues fully exposed to the solvent, that does not hinder the binding of RI. Similar results have been obtained for a third mutant of the pancreatic enzyme, engineered with the hinge peptide sequence of the seminal enzyme (residues 16-22) and the two cysteines in position 31 and 32, but lacking the hydrophobic Leu residue in position 28. The comparison of these three structures with those previously reported for other ribonuclease swapped dimers strongly suggests that, in addition to Pro19 and Leu28, the presence of a glycine at the N-terminal end of the hinge peptide is also important to push the swapped form of RNase A dimer into the compact quaternary organization observed for NCD-BS.

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Section: Discussionmentioning

confidence: 92%

Section: Recombinant Dna Methodologies and Protein Sample Preparationmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Toward an antitumor form of bovine pancreatic ribonuclease: The crystal structure of three noncovalent dimeric mutants

et al. 2009

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“…In particular, in both variants several hinge loop residues and/or others belonging to or interacting with the swappable N-terminal or C terminal domains have been mutated. 77,78,190,191 Many of them have been found to be key residues in stabilizing the domain-swapped oligomers, while others were found to promote oligomer dissociation towards the native proteins, i.e., monomeric RNaseA or dimeric BS-RNase. 190 Other factors which affect the stability of oligomers are pH, temperature, ionic strength of the medium, and the protein concentration.…”

Section: Bioactivity and Stability Of Protein Oligomersmentioning

confidence: 98%

A review on protein oligomerization process

Liu

2015

Int. J. Precis. Eng. Manuf.

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Enzymes or proteins are commonly present in oligomeric forms for active biological functions. The formation of protein oligomers, either in nature or by artificial means, can take place via covalent bonding or through weak-bond-network associations. Disulfide bonds are more common in forming covalent protein oligomers. Covalent bound protein oligomers usually have additional elements introduced, while proteins associated through weak-bond-networks usually do not involve any additional bound chemical groups. Proteins are commonly present in stable forms or folds. Oligomerization can occur when stable proteins unfold, creating exposed interfaces for association interactions. One well-known process of weak-bond-network oligomerization is domain swapping (DS). Separating the two touching/interacting domains creates opportunities for similar interactions with different protein molecules, leading to oligomerization. Both disulfide bonding and DS oligomerization can be dynamic (or reversible) at least at low Degree of Polymerizations (DPs). The dynamic nature of the protein oligomerization is important for bioactivity control. The morpheein model and the polymorph model are thus important in quantifying enzyme catalyzed biotransformations. Disulfide bonding and DS can act together to form supramolecules. The formation of supramolecules, such as amyloids, in nature can be benign or harmful. Industrial applications of protein oligomerization exploit the special functions /properties of the resultant supramolecules, such as multiple biofunctions, niche structure, etc..

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Stability strengths and weaknesses in protein structures detected by statistical potentials: Application to bovine seminal ribonuclease

2015

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We present an in silico method to estimate the contribution of each residue in a protein to its overall stability using three database-derived statistical potentials that are based on inter-residue distances, backbone torsion angles and solvent accessibility, respectively. Residues that contribute very unfavorably to the folding free energy are defined as stability weaknesses, whereas residues that show a highly stabilizing contribution are called stability strengths. Strengths and/or weaknesses on residues that are in spatial contact are clustered into 3-dimensional (3D) stability patches. The identification and analysis of strength- and weakness-containing regions in a protein may reveal structural or functional characteristics, and/or interesting spots to introduce mutations. To illustrate the power of our method, we apply it to bovine seminal ribonuclease. This enzyme catalyzes the degradation of RNA strands, and has the peculiarity of undergoing 3D domain swapping in physiological conditions. The weaknesses and strengths were compared among the monomeric, dimeric and swapped dimeric forms. We identified weaknesses among the catalytic residues and a mixture of weaknesses and strengths among the substrate-binding residues in the three forms. In the regions involved in 3D swapping, we observed an accumulation of weaknesses in the monomer, which disappear in the dimer and especially in the swapped dimer. Moreover, monomeric homologous proteins were found to exhibit less weaknesses in these regions, whereas mutants known to favor unswapped dimerization appear stabilized in this form. Our method has several perspectives for functional annotation, rational prediction of targeted mutations, and mapping of stability changes upon conformational rearrangements.

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Comparison of the structural and functional properties of RNase A and BS‐RNase: A stepwise mutagenesis approach

Cited by 26 publications

References 33 publications

Toward an antitumor form of bovine pancreatic ribonuclease: The crystal structure of three noncovalent dimeric mutants

Toward an antitumor form of bovine pancreatic ribonuclease: The crystal structure of three noncovalent dimeric mutants

A review on protein oligomerization process

Stability strengths and weaknesses in protein structures detected by statistical potentials: Application to bovine seminal ribonuclease

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