The structure of the complex of ribonuclease s with fluoride analogue of UpA at 2.5 Å resolution

Pavlovsky, A.G.; Borisova, S.N.; Borisov, V.V.; Antonov, Ilja V.; Karpeisky, M.Ya.

doi:10.1016/0014-5793(78)80766-2

Cited by 44 publications

(24 citation statements)

References 8 publications

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“…From the phenylalanines in RNase, Phe-120 is most directly involved in the binding of active-site inhibitors 11,34,35]. Therefore, we tentatively assign the phenylalanine resonance that is most strongly affected by inhibitors, F 3, to Phe-120.…”

Section: Efsects Of Active-site Inhibitors: Assignment O F F 3 ; Resomentioning

confidence: 91%

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by ¹H Nuclear Magnetic Resonance

Lenstra

Bolscher

Stob

et al. 1979

European Journal of Biochemistry

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1. The aromatic proton resonances in the 360-MHz 'H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments.2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-1 19) and two tyrosines are accessible to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119.3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-1 15 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120.5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180" about their CP-Cc" bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed.6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.

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Section: Efsects Of Active-site Inhibitors: Assignment O F F 3 ; Resomentioning

confidence: 91%

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by ¹H Nuclear Magnetic Resonance

Lenstra

Bolscher

Stob

et al. 1979

European Journal of Biochemistry

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“…These residues appear to be involved in substrate binding in HPR, similar to that in RNase A. In RNase A, Lys-7 is present in the phosphate binding subsite (38 -41), whereas Asn-71 and Glu-111 are present in the base binding subsite (41)(42)(43). The primary role of Gln-11, a conserved residue that donates a hydrogen bond to the reactive phosphoryl group of the bound substrate, is to prevent the nonproductive binding of the substrate (44).…”

Section: Cytotoxic Activity Of Hpr Mutantsmentioning

confidence: 97%

Interaction of Human Pancreatic Ribonuclease with Human Ribonuclease Inhibitor

Gaur¹,

Swaminathan²,

Batra

2001

Journal of Biological Chemistry

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Mammalian ribonucleases interact very strongly with the intracellular ribonuclease inhibitor (RI). Eukaryotic cells exposed to mammalian ribonucleases are protected from their cytotoxic action by the intracellular inhibition of ribonucleases by RI. Human pancreatic ribonuclease (HPR) is structurally and functionally very similar to bovine RNase A and interacts with human RI with a high affinity. In the current study, we have investigated the involvement of Lys-7, Gln-11, Asn-71, Asn-88, Gly-89, Ser-90, and Glu-111 in HPR in its interaction with human ribonuclease inhibitor. These contact residues were mutated either individually or in combination to generate mutants K7A, Q11A, N71A, E111A, N88R, G89R, S90R, K7A/E111A, Q11A/E111A, N71A/E111A, K7A/ N71A/E111A, Q11A/N71A/E111A, and K7A/Q11A/N71A/ E111A. Out of these, eight mutants, K7A, Q11A, N71A, S90R, E111A, Q11A/E111A, N71A/E111A, and K7A/N71A/ E111A, showed an ability to evade RI more than the wild type HPR, with the triple mutant K7A/N71A/E111A having the maximum RI resistance. As a result, these variants exhibited higher cytotoxic activity than wild type HPR. The mutation of Gly-89 in HPR produced no change in the sensitivity of HPR for RI, whereas it has been reported that mutating the equivalent residue Gly-88 in RNase A yielded a variant with increased RI resistance and cytotoxicity. Hence, despite its considerable homology with RNase A, HPR shows differences in its interaction with RI. We demonstrate that interaction between human pancreatic ribonuclease and RI can be disrupted by mutating residues that are involved in HPR-RI binding. The inhibitor-resistant cytotoxic HPR mutants should be useful in developing therapeutic molecules.

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“…In order to compare the kinetic data with structural information, there is a need for high-resolution structures of dinucleotide complexes of RNase A. 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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The structure of the complex of ribonuclease s with fluoride analogue of UpA at 2.5 Å resolution

Cited by 44 publications

References 8 publications

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by ¹H Nuclear Magnetic Resonance

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by ¹H Nuclear Magnetic Resonance

Interaction of Human Pancreatic Ribonuclease with Human Ribonuclease Inhibitor

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

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The structure of the complex of ribonuclease s with fluoride analogue of UpA at 2.5 Å resolution

Cited by 44 publications

References 8 publications

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by 1H Nuclear Magnetic Resonance

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by 1H Nuclear Magnetic Resonance

Interaction of Human Pancreatic Ribonuclease with Human Ribonuclease Inhibitor

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

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The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by ¹H Nuclear Magnetic Resonance

The Aromatic Residues of Bovine Pancreatic Ribonuclease Studied by ¹H Nuclear Magnetic Resonance