6FRG

Crystal structure of G-1F mutant of Ssp DnaB Mini-Intein variant M86


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.53 Å
  • R-Value Free: 0.170 
  • R-Value Work: 0.143 
  • R-Value Observed: 0.144 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine.

Friedel, K.Popp, M.A.Matern, J.C.J.Gazdag, E.M.Thiel, I.V.Volkmann, G.Blankenfeldt, W.Mootz, H.D.

(2019) Chem Sci 10: 239-251

  • DOI: https://doi.org/10.1039/c8sc01074a
  • Primary Citation of Related Structures:  
    6FRE, 6FRG, 6FRH

  • PubMed Abstract: 

    Inteins remove themselves from a precursor protein by protein splicing. Due to the concomitant structural changes of the host protein, this self-processing reaction has enabled many applications in protein biotechnology and chemical biology. We show that the evolved M86 mutant of the Ssp DnaB intein displays a significantly improved tolerance towards non-native amino acids at the N-terminally flanking (-1) extein position compared to the parent intein, in the form of both an artificially trans -splicing split intein and the cis -splicing mini-intein. Surprisingly, side chains with increased steric bulk compared to the native Gly(-1) residue, including d-amino acids, were found to compensate for the essential block B histidine in His73Ala mutants in the initial N-S acyl shift of the protein splicing pathway. In the case of the M86 intein, large (-1) side chains can even rescue protein splicing activity as a whole. With the comparison of three crystal structures, namely of the M86 intein as well as of its Gly(-1)Phe and Gly(-1)Phe/His73Ala mutants, our data supports a model in which the intein's active site can exert a strain by varying mechanisms on the different angles of the scissile bond at the extein-intein junction to effect a ground-state destabilization. The compensatory mechanism of the block B histidine is the first example for the direct functional role of an extein residue in protein splicing. It sheds new light on the extein-intein interplay and on possible consequences of their co-evolution as well as on the laboratory engineering of improved inteins.


  • Organizational Affiliation

    Institute of Biochemistry , University of Muenster , Wilhelm-Klemm-Str. 2 , 48149 Münster , Germany . Email: Henning.Mootz@uni-muenster.de.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Replicative DNA helicase169Synechocystis sp. PCC 6803 substr. KazusaMutation(s): 10 
Gene Names: dnaBslr0833
EC: 3.6.4.12
UniProt
Find proteins for Q55418 (Synechocystis sp. (strain PCC 6803 / Kazusa))
Explore Q55418 
Go to UniProtKB:  Q55418
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ55418
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.53 Å
  • R-Value Free: 0.170 
  • R-Value Work: 0.143 
  • R-Value Observed: 0.144 
  • Space Group: P 42 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 71.472α = 90
b = 71.472β = 90
c = 87.871γ = 90
Software Package:
Software NamePurpose
XDSdata reduction
Aimlessdata scaling
PHASERphasing
PHENIXrefinement
PDB_EXTRACTdata extraction
Cootmodel building

Structure Validation

View Full Validation Report



Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
German Research FoundationGermanyMO1073/3-2

Revision History  (Full details and data files)

  • Version 1.0: 2019-01-30
    Type: Initial release
  • Version 1.1: 2019-02-13
    Changes: Data collection, Database references
  • Version 1.2: 2019-02-20
    Changes: Data collection, Structure summary
  • Version 1.3: 2024-01-17
    Changes: Data collection, Database references, Refinement description