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2b5r
From Proteopedia
| 2b5r, resolution 1.65Å () | |||||||||
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| Gene: | bla (Escherichia coli) | ||||||||
| Activity: | Beta-lactamase, with EC number 3.5.2.6 | ||||||||
| Domains: | PenP, BLIP | ||||||||
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| Resources: | FirstGlance, OCA, PDBsum, RCSB, TOPSAN | ||||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||||
TEM1-β-Lactamase/ β-Lactamase Inhibitor Protein (BLIP)
Proteins bind one another in aqua's solution to form tight and specific complexes. Previously we have shown that this is achieved through the modular architecture of the interaction network formed by the interface residues, where tight cooperative interactions are found within modules but not between them. Here we extend this study to cover the entire interface of TEM1 beta-lactamase and its protein inhibitor BLIP using an improved method for deriving interaction maps based on REDUCE to add hydrogen atoms and then by evaluating the interactions using modifications of the programs PROBE, NCI and PARE. An extensive mutagenesis study of the interface residues indeed showed that each module is energetically independent on other modules, and that cooperativity is found only within a module. By solving the X-ray structure of two interface mutations affecting two different modules, we demonstrated that protein-protein binding occur via the structural reorganization of the binding modules, either by a "lock and key" or an induced fit mechanism. To explain the cooperativity within a module, we performed multiple-mutant cycle analysis of cluster 2 resulting in a high-resolution energy map of this module. Mutant studies are usually done in reference to alanine, which can be regarded as a deletion of a side-chain. However, from a biological perspective, there is a major interest to understand non-Ala substitutions, as they are most common. Using X-ray crystallography and multiple-mutant cycle analysis we demonstrated the added complexity in understanding non-Ala mutations. Here, a double mutation replacing the wild-type Glu,Tyr to Tyr,Asn on TEM1 (res id 104,105) caused a major backbone structural rearrangement of BLIP, changing the composition of two modules but not of other modules within the interface. This shows the robustness of the modular approach, yet demonstrates the complexity of in silico protein design.
Binding hot spots in the TEM1-BLIP interface in light of its modular architecture., Reichmann D, Cohen M, Abramovich R, Dym O, Lim D, Strynadka NC, Schreiber G, J Mol Biol. 2007 Jan 19;365(3):663-79. Epub 2006 Oct 3. PMID:17070843
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
The enzyme TEM1 and its protein inhibitor, form a . The complex where BLIP residues of the bound structure are colored blue and of the unbound are in cyan. TEM1 residues from the bound complex are in red and from the unbound structure in yellow. BLIP and TEM1 residues are labeled blue and red. The between BLIP and TEM1 was divided into six interface clusters (, , , , , ). Superpositions of these clusters from TEM1–BLIP (complex-1jtg), TEM1 (unbound-1btl) and BLIP (unbound, Strynadka et al, 1994) structures are shown. of cluster C1 from TEM1-BLIP wt, mutant and unbound structures (TEM1wt-BLIPwt complex, TEM1wt-BLIPD49A, unbound TEM1 (yellow), and unbound BLIP). D49 in BLIP is located in the center of C1, surrounded by 4 TEM1 residues. The D49A mutation (i.e. removal of a side chain) does not cause structural change in the TEM1-BLIP complex.
To analyze the contribution of non-alanine mutations, E104Y and Y105N in the TEM1 protein were constructed. These residues are polar and have a similar size, hence no major structural changes are expected for these mutants. The complex structure of TEM1 E104Y-Y105N (TEM1YN) with BLIPwt was solved. The YN mutation caused only small reduction of binding energy (4.2 kJ/mol), which is slightly less then that obtained for the alanine substitutions of these residues. The of TEM1wt-BLIPwt (yellow) complex (1jtg; TEM1 colored in magenta; BLIP in yellow) with TEM1YN (colored in lime) - BLIPwt (pink) complex. Only a (colored orange) in was observed. But, these small changes in TEM1 sequence lead to a major local of the between residues 46–53 in BLIP (yellow). This rearrangement of the loop is not observed in the unbound BLIP structure. This shows that the rearrangement of BLIP was caused by the TEM1 mutations, resulting in a new low energy state. of residues in wt and mutant complexes near the BLIP 46-53 loop. TEM1 wt E104 and Y105 are colored cyan, while mutant E104Y and Y105N are colored orange. BLIP residues colored in blue and red in the wt and mutant complexes.
About this Structure
2B5R is a Protein complex structure of sequences from Escherichia coli and Streptomyces clavuligerus. Full crystallographic information is available from OCA.
References
- Reichmann D, Cohen M, Abramovich R, Dym O, Lim D, Strynadka NC, Schreiber G. Binding hot spots in the TEM1-BLIP interface in light of its modular architecture. J Mol Biol. 2007 Jan 19;365(3):663-79. Epub 2006 Oct 3. PMID:17070843
- Strynadka NC, Jensen SE, Johns K, Blanchard H, Page M, Matagne A, Frere JM, James MN. Structural and kinetic characterization of a beta-lactamase-inhibitor protein. Nature. 1994 Apr 14;368(6472):657-60. PMID:8145854
Page seeded by OCA on Sun Jul 27 19:43:48 2008
