The Solution Structure and Dynamics of Human Pancreatic Ribonuclease Determined by NMR Spectroscopy Provide Insight into Its Remarkable Biological Activities and Inhibition

Kövér, Katalin E.; Bruix, Marta; Santoro, Jorge; Batta, Gyula; Laurents, Douglas V.; Rico, Manuel

doi:10.1016/j.jmb.2008.04.042

Cited by 26 publications

(14 citation statements)

References 69 publications

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“…This loop plays a remarkable role in the biological properties of BS-RNase because it includes the Asn-Gly sequence, which spontaneously deamidates under physiological conditions [29], and represents also a potential contact region with Ribonuclease Inhibitor [45], a horse-shoe shaped protein which readily and efficiently inactivates most pancreatic-like ribonucleases. Structural and relaxation data indicate that the mBS behaves like a compact, globular monomer, and the correlation time allowed us to exclude not only the presence of dimeric forms, which instead have been observed in the homologue human pancreatic protein [46], but also a partial opening of the protein. Moreover relaxation data highlight the presence of two main flexible regions, corresponding to the 16–22 and 65–72 loops; in addition, extra-mobility is found at the C-terminal region (112–115 loop and 119–121 strand), i.e.…”

Section: Discussionmentioning

confidence: 99%

“…For NMR dynamics, besides the conventional 15 N relaxation (T 1 , T 2 and 15 N-NOE) [69], the longitudinal and transverse 15 N and 1 H CSA/DD cross-correlated relaxation rates η zz ( 15 N), η xy ( 15 N), η xy ( 1 H) [46], were measured at 500.13 MHz on a Bruker DRX spectrometer. To this end, a series of 2D 1 H- 15 N HSQC spectra using sensitivity enhanced gradient pulse schemes [70] were recorded.…”

Section: Methodsmentioning

confidence: 99%

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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: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

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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“…34 The interaction of the phenolic O of Tyr33 and the charged N f of the catalytic Lys38 observed in most of the NMR solution structures could stabilize the charge on the latter and modulate the enzyme's activity. This interaction is neither observed in crystal structures of ECP with a bound substrate mimics (1H1H) 24 nor without (1QMT nor 1DYT) nor in the solution structures of the ECP homologs human RNase 1 (2k11) 35 nor bovine pancreatic RNase A (2AAS). 36 Interestingly, the Tyr33 side chain, which is located in the region involved in ECP binding to heparin 25 has been recently reported to be nitrated during eosinophil maturation.…”

Section: Discussionmentioning

confidence: 86%

The¹H,¹³C,¹⁵N resonance assignment, solution structure, and residue level stability of eosinophil cationic protein/RNase 3 determined by NMR spectroscopy

et al. 2009

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Eosinophil cationic protein (ECP)/human RNase 3, a member of the RNase A family, is a remarkably cytotoxic protein implicated in asthma and allergies. These activities are probably due to ECP's ability to interact with and disrupt membranes and depend on two Trp, 19 Arg, and possibly an extremely high conformational stability. Here, we have used NMR spectroscopy to assign essentially all (1)H, (15)N, and backbone (13)C resonances, to solve the 3D structure in aqueous solution and to quantify its residue-level stability. The NMR solution structure was determined on the basis of 2316 distance constraints and is well-defined (backbone RMSD = 0.81 A). The N-terminus and the loop composed of residues 114-123 are relatively well-ordered; in contrast, conformational diversity is observed for the loop segments 17-22, 65-68, and 92-95 and most exposed sidechains. The side chain NH groups of the two Trp and 19 Arg showed no significant protection against hydrogen/deuterium exchange. The most protected NH groups belong to the first and last two beta-strands, and curiously, the first alpha-helix. Analysis of their exchange rates reveals a strikingly high global stability of 11.8 kcal/mol. This value and other stability measurements are used to better quantify ECP's unfolding thermodynamics.

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“…The backbone 1 H and 15 N NMR assignments for human RNase 1 were determined previously [52]. We found that the presence of zwitterionic CTAB micelles elicited few changes in the 1 H and 15 N chemical shifts.…”

Section: Resultsmentioning

confidence: 61%

“…NMR spectra were acquired at 25°C with a Bruker Avance III 600 MHz spectrometer. 1 H, 15 N-HSQC NMR data were quantified and peak assignments were made with the program Sparky 3 (T. D. Goddard and D. G. Kneller, University of California, San Francisco) using the assignments determined from the solution structure of RNase 1 [52]. The vector changes of chemical shift (ΔΔ δ ) were determined with the equation:…”

Section: Methodsmentioning

confidence: 99%

Comparative functional analysis of ribonuclease 1 homologs: molecular insights into evolving vertebrate physiology

Lomax

Eller

Raines

2017

Biochemical Journal

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Pancreatic-type ribonucleases (ptRNases) comprise a class of highly conserved secretory endoribonucleases in vertebrates. The prototype of this enzyme family is ribonuclease 1 (RNase 1). Understanding the physiological roles of RNase 1 is becoming increasingly important, as engineered forms of the enzyme progress through clinical trials as chemotherapeutic agents for cancer. Here we present an in-depth biochemical characterization of RNase 1 homologs from a broad range of mammals (human, bat, squirrel, horse, cat, mouse, and cow) and non-mammalian species (chicken, lizard, and frog). We discover that the human homolog of RNase 1 has a pH optimum for catalysis, ability to degrade double-stranded RNA, and affinity for cell-surface glycans that are distinctly higher than those of its homologs. These attributes have relevance for human health. Moreover, the functional diversification of the ten RNase 1 homologs illuminates the regulation of extracellular RNA and other aspects of vertebrate evolution.

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The Solution Structure and Dynamics of Human Pancreatic Ribonuclease Determined by NMR Spectroscopy Provide Insight into Its Remarkable Biological Activities and Inhibition

Cited by 26 publications

References 69 publications

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

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

The¹H,¹³C,¹⁵N resonance assignment, solution structure, and residue level stability of eosinophil cationic protein/RNase 3 determined by NMR spectroscopy

Comparative functional analysis of ribonuclease 1 homologs: molecular insights into evolving vertebrate physiology

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The Solution Structure and Dynamics of Human Pancreatic Ribonuclease Determined by NMR Spectroscopy Provide Insight into Its Remarkable Biological Activities and Inhibition

Cited by 26 publications

References 69 publications

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

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

The1H,13C,15N resonance assignment, solution structure, and residue level stability of eosinophil cationic protein/RNase 3 determined by NMR spectroscopy

Comparative functional analysis of ribonuclease 1 homologs: molecular insights into evolving vertebrate physiology

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The¹H,¹³C,¹⁵N resonance assignment, solution structure, and residue level stability of eosinophil cationic protein/RNase 3 determined by NMR spectroscopy