The Buried Diversity of Bovine Seminal Ribonuclease: Shape and Cytotoxicity of the Swapped Non-covalent Form of the Enzyme

Merlino, Antonello; Ercole, Carmine; Picone, Delia; Pizzo, Elio; Mazzarella, L.; Sica, F.

doi:10.1016/j.jmb.2007.11.008

Cited by 32 publications

(49 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The electron density associated with the two hinge regions (residues [16][17][18][19][20][21][22] is well defined [ Fig. 1(C)].…”

Section: Overall Structurementioning

confidence: 99%

“…17 They differ for the swapping of the N-termini (residues 1-15) within a substantially identical quaternary framework. [18][19][20] In both isoforms, the dimeric interface is formed by the hinge peptides (residues [16][17][18][19][20][21][22] and the helices (residues [23][24][25][26][27][28][29][30][31][32][33][34] that comprise the four cysteines forming the two interchain disulfide bridges (Cys31-Cys32 0 and Cys32-Cys31 0 ) and the side chains of Leu28 and 28 0 that form stabilizing hydrophobic interactions across the molecular twofold axis. 18,20 In the most abundant swapped form, MxM-BSRNase, the dimeric interface also includes the closed interface arising from the swapped elements.…”

Section: Introductionmentioning

confidence: 99%

“…18,20 As a consequence of this latter contribution, MxM-BSRNase gives rise, upon selective reduction of the intersubunit disulfides, to a metastable noncovalent swapped dimer (NCD-BSRNase), whereas M¼M-BSRNase yields a monomeric derivative. 17 Although the two dimers present an almost undistinguishable external shape, 21 the cytotoxic action is a peculiar property of MxM-BSRNase. [13][14][15] Following the experimental results obtained in vitro, it is believed that in the reducing cytosolic compartment M¼M-BSRNase dissociates into monomers, which are strongly inhibited by RI, whereas MxM-BSRNase survives as NCD-BSRNase.…”

Section: Introductionmentioning

confidence: 99%

“…This dimer presents an overall structure highly reminiscent of the covalent forms, with the cysteine residues involved in the intersubunit linkages not far away from each other. 16,21 Because of its shape, it can easily evade interactions with RI. 16 In addition, the proximity of the cysteine residues may enhance the formation of the disulfide bridges and explain the higher stability of the covalent linkages in the swapped protein with respect to the unswapped one.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Structural features for the mechanism of antitumor action of a dimeric human pancreatic ribonuclease variant

et al. 2008

Self Cite

View full text Add to dashboard Cite

A specialized class of RNases shows a high cytotoxicity toward tumor cell lines, which is critically dependent on their ability to reach the cytosol and to evade the action of the ribonuclease inhibitor (RI). The cytotoxicity and antitumor activity of bovine seminal ribonuclease (BSRNase), which exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer, are peculiar properties of the swapped form. A dimeric variant (HHP2-RNase) of human pancreatic RNase, in which the enzyme has been engineered to reproduce the sequence of BSRNase helix-II (Gln28fiLeu, Arg31fiCys, Arg32fiCys, and Asn34fiLys) and to eliminate a negative charge on the surface (Glu111fiGly), is also extremely cytotoxic. Surprisingly, this activity is associated also to the unswapped form of the protein. The crystal structure reveals that on this molecule the hinge regions, which are highly disordered in the unswapped form of BSRNase, adopt a very well-defined conformation in both subunits. The results suggest that the two hinge peptides and the two Leu28 side chains may provide an anchorage to a transient noncovalent dimer, which maintains Cys31 and Cys32 of the two subunits in proximity, thus stabilizing a quaternary structure, similar to that found for the noncovalent swapped dimer of BSRNase, that allows the molecule to escape RI and/or to enhance the formation of the interchain disulfides.Keywords: crystal structure; antitumor agents; ribonuclease; 3D-domain swapping; dimers Abbreviations: BSRNase, bovine seminal ribonuclease; des(1-7)HP-RNase, human pancreatic ribonuclease in which the first seven residues have been deleted; HHP-RNase, covalent dimeric variant of human pancreatic ribonuclease in which four residues at the helix-II region are mutated according to BSRNase sequence (Gln28!Leu, Arg31!Cys, Arg32!Cys, and Asn34!Lys); HHP2-RNase, HHP-RNase with an additional mutation (Glu111!Gly); HP-RI, structure of human pancreatic ribonuclease complexed with ribonuclease inhibitor; HP-RNase, human pancreatic ribonuclease; MxM-BSRNase, dimeric form of BSRNase in which the chains swap their N-terminal tails; M¼M-BSRNase, unswapped dimer of BSRNase; M¼M-HHP2, unswapped dimer of HHP2-RNase; NCD-BSRNase, noncovalent swapped form of BSRNase; ONC, Onconase; PDB, Protein Data Bank; PM7, human pancreatic ribonuclease in which residues 1-21 are replaced by the corresponding residues of BSRNase; PM8, N-terminal swapped dimer of a variant of human pancreatic ribonuclease; RI, ribonuclease inhibitor; RMSD, root-mean-square deviation.Grant sponsor: Ministero dell'Istruzione, dell'Università e della Ricerca; Grant number: FIRB RBNE03B8KK and PRIN2007.

show abstract

“…The electron density associated with the two hinge regions (residues [16][17][18][19][20][21][22] is well defined [ Fig. 1(C)].…”

Section: Overall Structurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Structural features for the mechanism of antitumor action of a dimeric human pancreatic ribonuclease variant

et al. 2008

Self Cite

View full text Add to dashboard Cite

show abstract

“…26,27 It is generally accepted that the cytoxicity of some RNases is mainly related to their ability to evade the cytoplasmic RNase inhibitor (RI), so that they can freely act on cellular RNA without regulation. 28 The differences in the biological properties of BSRNase isomers have been explained considering that (a) in the reducing environment of the cytosol the two interchain disulfides are broken, (b) following the disruption of the 4CR motif, M5M dissociates into monomers, whereas M3M forms a metastable noncovalent dimeric intermediate (NCD) stabilized by the 3D-domain swapping, 22,27,29,30 and (c) the NCD species has been found by X-ray crystallography to adopt a quaternary structure that hinders the association with the proteic inhibitor. 30 This explanation of the cytotoxic activity of the swapped form of the seminal enzyme is consistent with the results of several studies carried out on ad hoc designed mutants, 24,31 of the seminal enzyme as well as on those of homologous monomeric pancreatic RNases.…”

Section: The Proteins Embodying the 4cr Motifmentioning

confidence: 99%

The four cysteines ring motif in proteins

Zagari¹

2009

Biopolymers

View full text Add to dashboard Cite

Disulfide bonds play fundamental roles in proteins. This work is devoted to highly rare motifs containing disulfide bonds. A search for four cysteines, forming a 16-atom membered ring (4CR) embodying two disulfide bonds, was carried out against all entries in the Protein Data Bank. Searching the crystallographic subset, only few protein molecules, all dimeric, were found to embody this peculiar structural feature, which establishes a covalent link between two different polypeptide chains. In contrast, in a peptide studied in solution by NMR, the four cysteines moiety includes only residues from one chain. A comparative analysis provided evidence for similarity and difference. It emerged that 4CR motif is highly rare and may serve to gain a specialized function.

show abstract

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

et al. 2009

View full text Add to dashboard Cite

The original structure of bovine seminal ribonuclease (BS-RNase), solved in 1993, represents a milestone in the story of protein structure, because it represented the first X-ray structure showing two polypeptide chains entangled through their terminal regions. It is generally assumed that this structural feature is the basis of several special biological activities, including a potent antitumor activity, but this has not been yet definitely proved. To assess this hypothesis, in this article we have analyzed the effects of the N-terminal hinge region and/or of Arg80 on the swapping propensity and cytotoxicity in newly designed proteins, using a covalent dimeric variant of bovine pancreatic ribonuclease (RNase A) as scaffold. All the proteins have a very poor cytotoxic activity, independently on the swapping propensity, that can even reach the same value of native BS-RNase. Overall our data suggest that the swapping represents still an essential requisite for the cytotoxic activity, because it keeps the dimeric structure stable even in the reducing cytosolic environment, but other features are essential to design dimeric antitumor ribonucleases, including a strong positive potential at the N-terminal face and a quaternary structure able to evade the cytosolic ribonuclease inhibitor, with or without the interchain disulfide bridges.

show abstract

The Buried Diversity of Bovine Seminal Ribonuclease: Shape and Cytotoxicity of the Swapped Non-covalent Form of the Enzyme

Cited by 32 publications

References 44 publications

Structural features for the mechanism of antitumor action of a dimeric human pancreatic ribonuclease variant

Structural features for the mechanism of antitumor action of a dimeric human pancreatic ribonuclease variant

The four cysteines ring motif in proteins

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

Contact Info

Product

Resources

About