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

Abstract

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.

Structural and functional importance of local and global conformational fluctuations in the RNase A superfamily

“…Nevertheless, motions for loop 4, Lys31 (Lys41) and His97 (His119), and conformational changes in the V 1 and V 2 domains, were also recently shown by crystallography to reflect those observed in structurally homologous RNases . Similar conclusions were drawn for monomeric forms of BS‐RNase, which appear to show analogous loop 4 flexibility and allosteric motions neighboring the distant loop 1 (residues 16–22) .…”

“…However, only a restricted set have been subjected to dynamic investigation, considerably restricting flexibility comparisons between evolutionarily diverse enzymes with this structural fold. Although this is not an entirely comprehensive list, experimental and/or theoretical dynamic studies have been performed on free, ligand‐bound, wild‐type and/or mutant forms of human and rat RNase 1 , human eosinophil‐derived neurotoxin (EDN, or RNase 2) , human ECP (or RNase 3) , human RNase 4 , bovine and human angiogenin (hANG, RNase 5) , the northern leopard frog ranpirnase (Onconase) , and monomeric derivatives of bovine seminal RNase (BS‐RNase) . The details of some of these dynamic studies will now be briefly described and compared with the local and global motional behavior of RNase A.…”

Influence of pH on the structure and stability of the sweet protein MNEI

“…This is likely linked to the sampling of a larger range of conformations, including precursors to unfolding. In order to extract k o and protection factors from the exchange rates, we first evaluated the effect of the pH on k ex, to assess the mechanism by which exchange occurs in the conditions employed for the kinetic measurements . It has been shown that under unimolecular exchange regime (EX1) there is no pH dependence of k ex whereas, in the bimolecular exchange limit (EX2), k ex will be proportional to pH .…”

Structure, stability, and IgE binding of the peach allergen Peamaclein (Pru p 7)

“…Spectral overlapping precluded the possibility of an unambiguous assignment of the other two cysteine pairs. Once a fully active recombinant sample is available a standard heteronuclear NMR study will be possible to evaluate in more detail the structural and dynamical features of the protein in solution, and to provide a more comprehensive mapping of the protein surface exposure, to highlight the potential epitopes eliciting the allergenic properties.…”