Trypsin Sheds Light on the Singular Case of Seminal RNase, a Dimer with Two Quaternary Conformations

Piccoli, Renata; Lorenzo, Claudia De; Piaz, Fabrizio Dal; Pucci, Piero; D’Alessio, Giuseppe

doi:10.1074/jbc.275.11.8000

Cited by 14 publications

(15 citation statements)

References 34 publications

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“…The replacement of A19P, E28L, K31C, and S32C on RNase A led to domain swapping and to the acquired biological properties (19). It was reported that cross-linked dimer and trimer of RNase A also exhibit these properties (39)(40)(41). It is unknown, however, if the cross-linked dimer is 3D domain-swapped and, if so, if it has the same structure as the unlinked RNase A dimer reported here.…”

Section: Discussionmentioning

confidence: 63%

The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1-Å resolution

Liu

Hart

Schlunegger

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

180

274

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The dimer of bovine pancreatic ribonuclease A (RNase A) discovered by Crestfield, Stein, and Moore in 1962 has been crystallized and its structure determined and refined to a 2.1-Å resolution. The dimer is 3D domainswapped. The N-terminal helix (residues 1-15) of each subunit is swapped into the major domain (residues 23-124) of the other subunit. The dimer of bull seminal ribonuclease (BSRNase) is also known to be domain-swapped, but the relationship of the subunits within the two dimers is strikingly different. In the RNase A dimer, the 3-stranded beta sheets of the two subunits are hydrogen-bonded at their edges to form a continuous 6-stranded sheet across the dimer interface; in the BS-RNase dimer, it is instead the two helices that abut. Whereas the BS-RNase dimer has 2-fold molecular symmetry, the two subunits of the RNase A dimer are related by a rotation of ϳ160°. Taken together, these structures show that intersubunit adhesion comes mainly from the swapped helical domain binding to the other subunit in the ''closed interface'' but that the overall architecture of the domain-swapped oligomer depends on the interactions in the second type of interface, the ''open interface.'' The RNase A dimer crystals take up the dye Congo Red, but the structure of a Congo Red-stained crystal reveals no bound dye molecule. Dimer formation is inhibited by excess amounts of the swapped helical domain. The possible implications for amyloid formation are discussed.

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Section: Discussionmentioning

confidence: 63%

The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1-Å resolution

Liu

Hart

Schlunegger

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

180

274

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“…From these experiments, we conclude that the nonhyperbolic kinetics observed with the unmodified enzyme are due to the interaction of the substrate with the different subsites of the enzyme rather than to different pre-existing conformations of RNase A (4) or to RNase aggregation (6). This hypothesis is discussed in terms of the configuration of the several binding subsites in RNase A, which is fundamental in relation to the physiological function of the enzyme, i.e.…”

mentioning

confidence: 88%

“…It is well known that RNase A can be split by controlled digestion with subtilisin yielding two fragments, the S-peptide (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and S-protein (residues 21-124), which can be separated easily (28). The separate fragments are inactive.…”

Section: The Substrate Concentration Dependence Of the Reaction Of Rnmentioning

confidence: 99%

The Subsites Structure of Bovine Pancreatic Ribonuclease A Accounts for the Abnormal Kinetic Behavior with Cytidine 2′,3′-Cyclic Phosphate

Moussaoui

Nogués

Guasch

et al. 1998

Journal of Biological Chemistry

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The kinetics of the hydrolysis of cytidine 2,3-cyclic phosphate (C>p) to 3-CMP by ribonuclease A are multiphasic at high substrate concentrations. We have investigated these kinetics by determining 3-CMP formation both spectrophotometrically and by a highly specific and quantitative chemical sampling method. With the use of RNase A derivatives that lack a functional p 2 binding subsite, evidence is presented that the abnormal kinetics with the native enzyme are caused by the sequential binding of the substrate to the several subsites that make up the active site of ribonuclease. The evidence is based on the following points. 1) Some of the unusual features found with native RNase A and C>p as substrate disappear when the derivatives lacking a functional p 2 binding subsite are used. 2) The k cat /K m values with oligocytidylic acids of increasing lengths (ending in C>p) show a behavior that parallels the specific velocity with C>p at high concentrations: increase in going from the monomer to the trimer, a decrease from tetramer to hexamer, and then an increase in going to poly(C). 3) Adenosine increases the k cat obtained with a fixed concentration of C>p as substrate. 4) High concentrations of C>p protect the enzyme from digestion with subtilisin, which results in a more compact molecule, implying large substrate concentration-induced conformational changes. The data for the hydrolysis of C>p by RNase A can be fitted to a fifth order polynomial that has been derived from a kinetic scheme based on the sequential binding of several monomeric substrate molecules.Bovine pancreatic RNase A (EC 3.1.27.5) catalyzes the breakdown of RNA by means of a transesterification from the 5Ј position of one nucleotide to the 2Ј position of the adjacent nucleotide with the formation of a 2Ј,3Ј-cyclic phosphate product (Scheme 1). In an aqueous environment, the cyclic nucleotide is irreversibly hydrolyzed to a 3Ј nucleotide. The forward direction of the reaction requires a dinucleoside monophosphate as the minimum size for the substrate, whereas the reverse direction of the reaction can take place with pyrimidine 2Ј,3Ј-cyclic mononucleotides. It appears that the hydrolytic step is just a special case of the reverse transphosphorylation, as has been emphasized by Cuchillo et al. (1,2) and by Thompson et al. (3).Several kinetic studies with RNase A have been carried out following the hydrolysis of either uridine or cytidine 2Ј,3Ј-cyclic phosphate as substrates. At relatively low concentrations of this type of substrate, in an aqueous environment, the only significant route is that of hydrolysis, and a hyperbolic substrate concentration dependence is usually obtained. However, working at high substrate concentrations, Walker et al. (4,5) observed that at pH 7.0, the dependence of the rate of hydrolysis with substrate concentration shows a nonhyperbolic behavior: there is a drop at about 25 mM to 30 mM CϾp 1 followed by a second rise and then a gradual decline, which they interpreted by an allosteric model in which there is a substra...

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“…Consequently, when these experiments are performed using a series of proteases with different specificity under conditions that favors a single bond cleavage (complementary proteolysis), the pattern of preferred cleavage sites will depict the exposed regions in the protein molecule (14,15). This strategy was also employed to investigate conformational changes occurring in protein structure under different experimental conditions (16 -19) or during quaternary forms interchange (20,21), as well as for the definition of the interface regions in protein complexes (22)(23)(24).…”

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confidence: 99%

Structural Characterization of the M* Partly Folded Intermediate of Wild Type and P138A Aspartate Aminotransferase from Escherichia coli

Birolo¹,

Piaz²,

Pucci³

et al. 2002

Journal of Biological Chemistry

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A combination of spectroscopic techniques, hydrogen/ deuterium exchange, and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the topology of the monomeric M* partly folded intermediate of aspartate aminotransferase from Escherichia coli in wild type (WT) as well as in a mutant form in which the highly conserved cis-proline at position 138 was replaced by a trans-alanine (P138A). Fluorescence analysis indicates that, although M* is an off-pathway intermediate in the folding of WT aspartate aminotransferase from E. coli, it seems to coincide with an onpathway folding intermediate for the P138A mutant. Spectroscopic data, hydrogen/deuterium exchange, and limited proteolysis experiments demonstrated the occurrence of conformational differences between the two M* intermediates, with P138A-M* being conceivably more compact than WT-M*. Limited proteolysis data suggested that these conformational differences might be related to a different relative orientation of the small and large domains of the protein induced by the presence of the cis-proline residue at position 138. These differences between the two M* species indicated that in WT-M* Pro138 is in the cis conformation at this stage of the folding process. Moreover, hydrogen/deuterium exchange results showed the occurrence of few differences in the native N 2 forms of WT and P138A, the spectroscopic features and crystallographic structures of which are almost superimposable.The mechanism by which proteins fold into their unique native structures is still a central problem in structural biology. It is increasingly recognized that the structure of non-native states of proteins can provide significant insight into fundamental issues such as the relationship between protein sequences and three-dimensional structures, the nature of protein folding pathways, the stability of proteins and their turnover in the cell, and the transport of proteins across membranes (1). Moreover, intermediate states experienced by proteins in vivo often play a major role in protein association and aggregation, leading to the "so-called" conformational diseases (2). In contrast to the large amount of structural information available on native folded proteins, however, too few partly folded intermediates have been characterized thoroughly enough to propose general models on how the native state is attained and which is the structure of transient folding states. Investigation of a wide range of non-native states would then be of considerable value. To meet this need, new analytical strategies able to characterize transient species and to define the molecular details through which diverse proteins fold are required.Recently, structural biologists have turned their attention to integrated strategies for the definition of the surface topology of proteins and protein complexes. Although these approaches provide low resolution data, they are amenable to the analysis of transient species and partly folded intermediates. Limited proteolysis and amide hydrogen ex...

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Trypsin Sheds Light on the Singular Case of Seminal RNase, a Dimer with Two Quaternary Conformations

Cited by 14 publications

References 34 publications

The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1-Å resolution

The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1-Å resolution

The Subsites Structure of Bovine Pancreatic Ribonuclease A Accounts for the Abnormal Kinetic Behavior with Cytidine 2′,3′-Cyclic Phosphate

Structural Characterization of the M* Partly Folded Intermediate of Wild Type and P138A Aspartate Aminotransferase from Escherichia coli

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