2P0X

solution structure of a non-biological ATP-binding protein


Experimental Data Snapshot

  • Method: SOLUTION NMR
  • Conformers Calculated: 200 
  • Conformers Submitted: 20 
  • Selection Criteria: structures with the lowest energy 

wwPDB Validation   3D Report Full Report


This is version 1.1 of the entry. See complete history


Literature

Structure and Evolutionary Analysis of a Non-biological ATP-binding Protein

Mansy, S.S.Zhang, J.Kummerle, R.Nilsson, M.Chou, J.J.Szostak, J.W.Chaput, J.C.

(2007) J Mol Biol 371: 501-513

  • DOI: https://doi.org/10.1016/j.jmb.2007.05.062
  • Primary Citation of Related Structures:  
    2P0X

  • PubMed Abstract: 

    We present a structural and functional analysis of the evolutionary optimization of a non-biological protein derived from a library of random amino acid sequences. A series of previously described in vitro selection experiments transformed a low-affinity ancestral sequence into a stably folded, high affinity ATP binding protein structure. While the evolutionarily optimized protein differs from its ancestral sequence through the accumulation of 12 amino acid mutations, the means by which those mutations enhance the stability and functionality of the protein were not well understood. We used a combination of mutagenesis, biochemistry, and NMR spectroscopy to investigate the structural and functional significance of each mutation. We solved the three-dimensional structure of the folding optimized protein by solution NMR, which revealed a fourth strand of the beta-sheet of the alpha/beta-fold that was not observed in an earlier crystallographic analysis of a less stable version of the protein. The structural rigidity of the newly identified beta-strand was confirmed by T1, T2, and heteronuclear nuclear Overhauser enhancement (NOE) measurements. Biochemical experiments were used to examine point mutations that revert the optimized protein back to the ancestral residue at each of the 12 sites. A combination of structural and functional data was then used to interpret the significance of each amino acid mutation. The enhanced ATP affinity was largely due to the emergence of a patch of positive charge density on the protein surface, while the increased solubility resulted from several mutations that increased the hydrophilicity of the protein surface, thereby decreasing protein aggregation. One mutation may stabilize the hydrophobic face of the beta-sheet.


  • Organizational Affiliation

    Howard Hughes Medical Institute, and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
abiotic ATP-binding, folding optimized protein64synthetic geneMutation(s): 0 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
ATP
Query on ATP

Download Ideal Coordinates CCD File 
C [auth A]ADENOSINE-5'-TRIPHOSPHATE
C10 H16 N5 O13 P3
ZKHQWZAMYRWXGA-KQYNXXCUSA-N
ZN
Query on ZN

Download Ideal Coordinates CCD File 
B [auth A]ZINC ION
Zn
PTFCDOFLOPIGGS-UHFFFAOYSA-N
Binding Affinity Annotations 
IDSourceBinding Affinity
ATP PDBBind:  2P0X Kd: 494 (nM) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: SOLUTION NMR
  • Conformers Calculated: 200 
  • Conformers Submitted: 20 
  • Selection Criteria: structures with the lowest energy 

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2007-08-07
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Version format compliance