Novel non-productively bound ribonuclease inhibitor complexes — high resolution X-ray refinement studies on the binding of RNase-A to cytidylyl-2′,5′-guanosine (2′,5′CpG) and deoxycytidylyl-3′,5′-guanosine (3′,5′dCpdG)

Aguilar, Carlos Fernando; Thomas, P.J.; Moss, David S.; Mills, Alberto; Palmer, Rex A.

doi:10.1016/0167-4838(91)90435-3

Cited by 32 publications

(19 citation statements)

References 17 publications

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“…Indeed, in the ligand-free form, water molecules are located in the active site ( Figure 2B), whereas in lb-NCD-PLCC, dCpdG is positioned in the retrobinding mode similar to that observed for guanine containing dinucleotide complexed to RNase A [44][45][46] and BS-RNase. 47 The purine base is located in B1 1 and is hydrogen bonded to Thr45, the guanine ribose moiety is exposed to the solvent and does not interact with the protein, and the phosphate group is only partially visible.…”

Section: Ligands Bound At the Active Sitesmentioning

confidence: 83%

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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Section: Ligands Bound At the Active Sitesmentioning

confidence: 83%

Toward an antitumor form of bovine pancreatic ribonuclease: The crystal structure of three noncovalent dimeric mutants

et al. 2009

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“…The structure of RNase A complexed with d(CpA) was primarily determined in order to gain more detailed information on the interactions at the downstream binding site and to compare the binding mode to that found in the crystal structures of RNase A soaked with 2',5'-CpG and d(CpG) (Aguilar et al, 1991(Aguilar et al, , 1992. In those last 2 high-resolution structures, the downstream guanine was bound in the major binding site, and the active site was occupied by an inorganic sulfate or phosphate anion.…”

Section: Discussionmentioning

confidence: 99%

“…Previously published structures of dinucleotide complexes of RNase A (UpcA, Richards & Wyckoff, 1973;2',5'-CpA, Wodak et al, 1977; 2'F-dUpA, Pavlovsky et al, 1978) were not determined at high resolution nor extensively refined. The recently published, highly refined structures of the complexes of RNase A with 2',5'-CpG and d(CpG) do not address this question because the inhibitors were found to be bound in a nonproductive way (Aguilar et al, 1991(Aguilar et al, , 1992.…”

Section: 'mentioning

confidence: 99%

The structures of rnase a complexed with 3′‐CMP and d(CpA): Active site conformation and conserved water molecules

et al. 1994

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The interactions of RNase A with cytidine 3'-monophosphate (3"CMP) and deoxycytidyl-3',5'-deoxyadenosine (d(CpA)) were analyzed by X-ray crystallography. The 3'-CMP complex and the native structure were determined from trigonal crystals, and the d(CpA) complex from monoclinic crystals. The differences between the overall structures are concentrated in loop regions and are relatively small. The protein-inhibitor contacts are interpreted in terms of the catalytic mechanism. The general base His 12 interacts with the 2' oxygen, as does the electrostatic catalyst Lys 41. The general acid His 119 has 2 conformations (A and B) in the native structure and is found in, respectively, the A and the B conformation in the d(CpA) and the 3'-CMP complex. From the present structures and from a comparison with RNase T1, we propose that His 119 is active in the A conformation. The structure of the d(CpA) complex permits a detailed analysis of the downstream binding site, which includes His 119 and Asn 71. The comparison of the present RNase A structures with an inhibitor complex of RNase T1 shows that there are important similarities in the active sites of these 2 enzymes, despite the absence of any sequence homology. The water molecules were analyzed in order to identify conserved water sites. Seventeen water sites were found to be conserved in RNase A structures from 5 different space groups. It is proposed that 7 of those water molecules play a role in the binding of the N-terminal helix to the rest of the protein and in the stabilization of the active site.

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“…Some show equal preference for cytosine and uracil, e.g. bovine RNase A, human pancreatic ribonuclease, bullfrog RC-RNase and RCRNase L1 (19,36,39). Others prefer uracil, e.g.…”

Section: N-terminal Pyroglutamate Involved In Bmentioning

confidence: 99%

“…The structure of ribonuclease-oligonucleotide complex has been solved, e.g. RNase A complexed with d(ATAAG), whereas retrobindings were observed in the crystal structure of RNase A complexed with dinucleotide, 2Ј,5Ј-UpG, 2Ј,5Ј-CpG, or 3Ј,5Ј-d(CpG) or in the solution structure of RC-RNase complexed with 2Ј,5Ј-CpG or 3Ј,5Ј-d(CpG) (17,(35)(36)(37). In retrobinding form, the orientation of substrate differs from that in the catalytic binding form.…”

Section: N-terminal Pyroglutamate Involved In Bmentioning

confidence: 99%

Residues Involved in the Catalysis, Base Specificity, and Cytotoxicity of Ribonuclease from Rana catesbeianaBased upon Mutagenesis and X-ray Crystallography

Leu¹,

Chern²,

Wang³

et al. 2003

Journal of Biological Chemistry

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The Rana catesbeiana (bullfrog) ribonucleases, which belong to the RNase A superfamily, exert cytotoxicity toward tumor cells. RC-RNase, the most active among frog ribonucleases, has a unique base preference for pyrimidine-guanine rather than pyrimidine-adenine in RNase A. Residues of RC-RNase involved in base specificity and catalytic activity were determined by sitedirected mutagenesis, k cat /K m analysis toward dinucleotides, and cleavage site analysis of RNA substrate. The results show that Pyr-1 (N-terminal pyroglutamate), Lys-9, and Asn-38 along with His-10, Lys-35, and His-103 are involved in catalytic activity, whereas Pyr-1, Thr-39, Thr-70, Lys-95, and Glu-97 are involved in base specificity. The cytotoxicity of RC-RNase is correlated, but not proportional to, its catalytic activity. The crystal structure of the RC-RNase⅐d(ACGA) complex was determined at 1.80 Å resolution. Residues Lys-9, His-10, Lys-35, and His-103 interacted directly with catalytic phosphate at the P 1 site, and Lys-9 was stabilized by hydrogen bonds contributed by Pyr-1, Tyr-28, and Asn-38. Thr-70 acts as a hydrogen bond donor for cytosine through Thr-39 and determines B 1 base specificity. Interestingly, Pyr-1 along with Lys-95 and Glu-97 form four hydrogen bonds with guanine at B 2 site and determine B 2 base specificity.Ribonucleases are found widely within living organisms and are thought to play an important role in the metabolism of RNA. Recently, it has been shown that several members of the bovine ribonuclease superfamily exhibit biological functions in addition to intrinsic ribonucleolytic activities. Human eosinophil-derived neurotoxin and eosinophil cationic protein exert neurotoxicity (1) as well as antiparasitic activity (2), human angiogenin induces blood vessel formation (3), and frog ribonuclease exhibits antitumor activity (4, 5). Ribonucleolytic activity is essential for the biological functions of these proteins (6 -12).Bovine pancreatic ribonuclease, known as RNase A, in the ribonuclease superfamily is well characterized and is a valuable model for the study of structure-function relationships and protein refolding (13,14). It consists of 124 amino acid residues linked with four pairs of disulfide bridges and possesses a substrate preference for pyrimidine-adenosine in the RNA sequence but no cytotoxicity toward tumor cells. There are three subsites within RNase A molecule: the P 1 site, at which phosphodiester bond cleavage occurs; the B 1 site, for binding pyrimidine, which donates oxygen via its ribose to the scissile bond; and the B 2 site, for binding the adenine ring on the opposite site of the scissile bond. Three amino acid residues, His-12, Lys-41, and His-119, at the P 1 site are involved in catalytic activity. Four amino acid residues, Thr-45, Asp-83, Phe-120, and Ser-123, at the B 1 site are involved in the binding of the 5Ј-ribonucleoside, pyrimidine, whereas two residues, Asn-71 and Glu-111, at the B 2 site are involved in the binding of the 3Ј-ribonucleoside, adenosine (14 -18).A new group of ribon...

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Novel non-productively bound ribonuclease inhibitor complexes — high resolution X-ray refinement studies on the binding of RNase-A to cytidylyl-2′,5′-guanosine (2′,5′CpG) and deoxycytidylyl-3′,5′-guanosine (3′,5′dCpdG)

Cited by 32 publications

References 17 publications

Toward an antitumor form of bovine pancreatic ribonuclease: The crystal structure of three noncovalent dimeric mutants

Toward an antitumor form of bovine pancreatic ribonuclease: The crystal structure of three noncovalent dimeric mutants

The structures of rnase a complexed with 3′‐CMP and d(CpA): Active site conformation and conserved water molecules

Residues Involved in the Catalysis, Base Specificity, and Cytotoxicity of Ribonuclease from Rana catesbeianaBased upon Mutagenesis and X-ray Crystallography

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