Database accession: MF7000848
Name: Bleomycin-binding protein, mutant (Streptoalloteichus hindustanus)
PDB ID: 1xrk
Experimental method: X-ray (1.50 Å)
Assembly: Homodimer
Source organism: Streptoalloteichus hindustanus
Primary publication of the structure:
Brouns SJ, Wu H, Akerboom J, Turnbull AP, de Vos WM, van der Oost J
Engineering a selectable marker for hyperthermophiles.
(2005) J. Biol. Chem. 280: 11422-31
PMID: 15640151
Abstract:
Limited thermostability of antibiotic resistance markers has restricted genetic research in the field of extremely thermophilic Archaea and bacteria. In this study, we used directed evolution and selection in the thermophilic bacterium Thermus thermophilus HB27 to find thermostable variants of a bleomycin-binding protein from the mesophilic bacterium Streptoalloteichus hindustanus. In a single selection round, we identified eight clones bearing five types of double mutated genes that provided T. thermophilus transformants with bleomycin resistance at 77 degrees C, while the wild-type gene could only do so up to 65 degrees C. Only six different amino acid positions were altered, three of which were glycine residues. All variant proteins were produced in Escherichia coli and analyzed biochemically for thermal stability and functionality at high temperature. A synthetic mutant resistance gene with low GC content was designed that combined four substitutions. The encoded protein showed up to 17 degrees C increased thermostability and unfolded at 85 degrees C in the absence of bleomycin, whereas in its presence the protein unfolded at 100 degrees C. Despite these highly thermophilic properties, this mutant was still able to function normally at mesophilic temperatures in vivo. The mutant protein was co-crystallized with bleomycin, and the structure of the binary complex was determined to a resolution of 1.5 A. Detailed structural analysis revealed possible molecular mechanisms of thermostabilization and enhanced antibiotic binding, which included the introduction of an intersubunit hydrogen bond network, improved hydrophobic packing of surface indentations, reduction of loop flexibility, and alpha-helix stabilization. The potential applicability of the thermostable selection marker is discussed.
Annotations from the GeneOntology database. Only terms that fit at least two of the interacting proteins are shown.Molecular function: not assigned
Biological process:
response to antibiotic response to antibiotic
Cellular component: not assigned
Structural annotations of the participating protein chains.Entry contents: 2 distinct polypeptide molecules
Chains: A, B
Notes: All chains according to the most probable oligomerization state stored in PDBe were considered.
Number of unique protein segments: 1
Name: Bleomycin resistance protein
Source organism: Streptoalloteichus hindustanus
Length: 124 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDDVTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGREFALRDPAGNCVHFVAEEQD
UniProtKB AC: P17493 (positions: 2-121)
Coverage: 96%
Name: Bleomycin resistance protein
Source organism: Streptoalloteichus hindustanus
Length: 124 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDDVTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGREFALRDPAGNCVHFVAEEQD
UniProtKB AC: P17493 (positions: 2-122)
Coverage: 97%
Evidence demonstrating that the participating proteins are unstructured prior to the interaction and their folding is coupled to binding. Representative domain in related structures: Glyoxalase superfamily
Evidence level: Indirect evidence
Evidence coverage: The full structure participates in mutual synergistic folding.
Complex Evidence:
The bleomycin resistance protein forms a tight dimer with a large hydrophobic interface and domain-swapping involving the N-terminal end of both monomers. Only dimeric form could be detected in solution by several dedicated methods. The antibiotic binds to he dimer interface contacting both monomers (PMID:7516875). Temperature-induced equilibrium unfolding experiments on the Shble protein and its engineered variants suggested a two-state model of unfolding (although unfolding was largely irreversible with only 40% of the native folded signal regained during the refolding phase) and the strong stabilizing effect of bleomycin on the dimer based on largely increased thermostability of the protein in the presence of the antibiotic (PMID:15640151).
Chain A:
N/A
Chain B:
N/A
Surface and contacts features:
Structures from the PDB that contain the same number of proteins, and the proteins from the two structures show a sufficient degree of pairwise similarity, i.e. they belong to the same UniRef90 cluster (the full proteins exhibit at least 90% sequence identity) and convey roughly the same region to their respective interactions (the two regions from the two proteins share a minimum of 70% overlap). Download the CIF file (.cif)
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