2GMK

Crystal structure of onconase double mutant with spontaneously-assembled (AMP) 4 stack


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.65 Å
  • R-Value Free: 0.217 
  • R-Value Work: 0.165 

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This is version 2.2 of the entry. See complete history


Literature

Structural basis for catalysis by onconase.

Lee, J.E.Bae, E.Bingman, C.A.Phillips Jr., G.N.Raines, R.T.

(2008) J Mol Biol 375: 165-177

  • DOI: https://doi.org/10.1016/j.jmb.2007.09.089
  • Primary Citation of Related Structures:  
    2GMK, 2I5S

  • PubMed Abstract: 

    Onconase (ONC) is a homolog of bovine pancreatic ribonuclease (RNase A) from the frog Rana pipiens. ONC displays antitumoral activity and is in advanced clinical trials for the treatment of cancer. Here, we report the first atomic structures of ONC-nucleic acid complexes: a T89N/E91A ONC-5'-AMP complex at 1.65 A resolution and a wild-type ONC-d(AUGA) complex at 1.90 A resolution. The latter structure and site-directed mutagenesis were used to reveal the atomic basis for substrate recognition and turnover by ONC. The residues in ONC that are proximal to the scissile phosphodiester bond (His10, Lys31, and His97) and uracil nucleobase (Thr35, Asp67, and Phe98) are conserved from RNase A and serve to generate a similar bell-shaped pH versus k(cat)/K(M) profile for RNA cleavage. Glu91 of ONC forms two hydrogen bonds with the guanine nucleobase in d(AUGA), and Thr89 is in close proximity to that nucleobase. Installing a neutral or cationic residue at position 91 or an asparagine residue at position 89 virtually eliminated the 10(2)-fold guanine:adenine preference of ONC. A variant that combined such substitutions, T89N/E91A ONC, actually preferred adenine over guanine. In contrast, installing an arginine residue at position 91 increased the guanine preference and afforded an ONC variant with the highest known k(cat)/K(M) value. These data indicate that ONC discriminates between guanine and adenine by using Coulombic interactions and a network of hydrogen bonds. The structure of the ONC-d(AUGA) complex was also used to probe other aspects of catalysis. For example, the T5R substitution, designed to create a favorable Coulombic interaction between ONC and a phosphoryl group in RNA, increased ribonucleolytic activity by twofold. No variant, however, was more toxic to human cancer cells than wild-type ONC. Together, these findings provide a cynosure for understanding catalysis of RNA cleavage in a system of high medicinal relevance.


  • Organizational Affiliation

    Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706-1544, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
P-30 protein104Lithobates pipiensMutation(s): 2 
Gene Names: RNP30_RANPI
EC: 3.1.27
UniProt
Find proteins for P22069 (Lithobates pipiens)
Explore P22069 
Go to UniProtKB:  P22069
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP22069
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
PCA
Query on PCA
A
L-PEPTIDE LINKINGC5 H7 N O3GLN
Binding Affinity Annotations 
IDSourceBinding Affinity
AMP PDBBind:  2GMK Ki: 5.90e+5 (nM) from 1 assay(s)
Binding MOAD:  2GMK Ki: 5.90e+5 (nM) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.65 Å
  • R-Value Free: 0.217 
  • R-Value Work: 0.165 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 28.994α = 90
b = 52.112β = 90
c = 66.142γ = 90
Software Package:
Software NamePurpose
SAINTdata scaling
MOLREPphasing
REFMACrefinement
PDB_EXTRACTdata extraction

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Revision History  (Full details and data files)

  • Version 1.0: 2006-04-25
    Type: Initial release
  • Version 1.1: 2008-01-15
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 2.0: 2019-12-25
    Changes: Database references, Derived calculations, Polymer sequence
  • Version 2.1: 2021-10-20
    Changes: Database references, Derived calculations
  • Version 2.2: 2023-08-30
    Changes: Data collection, Refinement description