The Role of the Hinge Loop in Domain Swapping

Picone, Delia; Fiore, Alberto; Ercole, Carmine; Franzese, M.; Sica, F.; Tomaselli, Simona; Mazzarella, L.

doi:10.1074/jbc.m413157200

Cited by 31 publications

(13 citation statements)

References 32 publications

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“…Hence in the case of BS-RNase the dislocation of the N-termini requires only a partial unfolding step associated with a small enthalpic barrier. As a consequence, for this protein the swapping is a physiological, equilibrium process [19] and the molar ratio between swapped and un-swapped forms depends on tiny structural differences that influence the energetic balance. Upon selective reduction of the interchain disulphide bridges, under mild conditions [20], the swapped form of BS-RNase retains a dimeric structure stabilized by non-covalent interactions, whereas the unswapped one is converted into a monomeric derivative, indicated henceforth as mBS.…”

Section: Introductionmentioning

confidence: 99%

NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D Domain Swapping

et al. 2012

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Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.

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Section: Introductionmentioning

confidence: 99%

NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D Domain Swapping

et al. 2012

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“…33 The swapped and unswapped forms (MxM and M5M) of PLCC and LCC were prepared as previously described. 26,34 The mutated protein, sequestered into inclusion bodies, was unfolded, purified, and refolded in the monomeric form, with Cys31 and 32 linked to two glutathione molecules. When the protecting glutathione moieties were removed from the expressed RNase mutants by mild reduction, the resulting free monomers readily formed dimers.…”

Section: Recombinant Dna Methodologies and Protein Sample Preparationmentioning

confidence: 99%

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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“…4,5 On the other hand, mutagenesis studies provide evidence that proline residues located in the hinge region can influence the rate of swapped dimer formation in some systems 6 but not in others. 7 The role of hinge prolines in promoting swapping has also been inferred from surveys of known protein crystal structures. 8 Furthermore, a strong case for the involvement of protein unfolding/unfolding in swapping has been made in studies on the cell cycle regulatory protein p13suc1.…”

Section: Introductionmentioning

confidence: 99%