Stabilization of GroEL minichaperones by core and surface mutations

Wang, Qinghua; Buckle, Ashley M.; Fersht, Alan R.

doi:10.1006/jmbi.2000.3716

Cited by 36 publications

(17 citation statements)

References 39 publications

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“…There is evidence to suggest that in many cases the evolution of proteins has occurred to optimize function at the expense of stability (61-64); for example, there are exquisite stereochemical requirements for catalysis, whereas protein stability requirements usually are less onerous (61)(62)(63)(64). A serpin from a thermophilic organism represents an interesting twist to this hypothesis, as the evolutionary selection pressure is complicated by the presence of two structurally and energetically unique folded states (65).…”

Section: Resultsmentioning

confidence: 99%

The High Resolution Crystal Structure of a Native Thermostable Serpin Reveals the Complex Mechanism Underpinning the Stressed to Relaxed Transition

Fulton

Buckle

Cabrita

et al. 2005

Journal of Biological Chemistry

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Serpins fold into a native metastable state and utilize a complex conformational change to inhibit target proteases. An undesirable result of this conformational flexibility is that most inhibitory serpins are heat sensitive, forming inactive polymers at elevated temperatures. However, the prokaryote serpin, thermopin, from Thermobifida fusca is able to function in a heated environment. We have determined the 1.8 Å x-ray crystal structure of thermopin in the native, inhibitory conformation. A structural comparison with the previously determined 1.5 Å structure of cleaved thermopin provides detailed insight into the complex mechanism of conformational change in serpins. Flexibility in the shutter region and electrostatic interactions at the top of the A ␤-sheet (the breach) involving the C-terminal tail, a unique structural feature of thermopin, are postulated to be important for controlling inhibitory activity and triggering conformational change, respectively, in the native state. Here we have discussed the structural basis of how this serpin reconciles the thermodynamic instability necessary for function with the stability required to withstand elevated temperatures.Serpins (peptidase inhibitor family I4) are the largest superfamily of protease inhibitors with over 800 members identified to date (1-4). A recent authoritative classification of known peptidase inhibitors reveals that serpins are the only inhibitor family present in all three superkingdoms of life (Eukarya, Bacteria, and Archaea) as well as certain viruses (1). The majority of serpins are found in multicellular eukaryotes and inhibit serine or (more rarely) cysteine proteases. Inhibitory serpins are unusual molecules that fold into a native metastable state and utilize a complex conformational change to achieve protease inhibition (4 -6). Upon docking with a target protease, a flexible exposed region termed the reactive center loop (RCL) 1 is cleaved and the region N-terminal to the site of proteolysis (termed P1-P15) 2 inserts into the center of the large A ␤-sheet, forming an additional ␤-strand and causing a large conformational change (termed the Stressed (S) to Relaxed (R) transition) throughout the molecule (8 -10). Following cleavage, and throughout the S to R transition, the protease remains covalently attached to the serpin via an acyl bond between the side chain of the active site serine (or cysteine) and the carbonyl oxygen of the P1 residue in the RCL. The x-ray crystal structure of antitrypsin in complex with trypsin revealed that the enzyme is translocated 75 Å to the base of the inhibitor where it is trapped in a distorted, inactive conformation (11-14). The events following successful protease inhibition are irreversible, and serpins are thus termed suicide inhibitors. The size and conformational flexibility of serpins allow for an exquisite degree of functional control, and many serpins require specific co-factors to effectively inhibit target enzymes. For example, the thrombin/factor Xa inhibitor antithrombin circulates in a rel...

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Section: Resultsmentioning

confidence: 99%

The High Resolution Crystal Structure of a Native Thermostable Serpin Reveals the Complex Mechanism Underpinning the Stressed to Relaxed Transition

Fulton

Buckle

Cabrita

et al. 2005

Journal of Biological Chemistry

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“…Indeed, this is a proven method of increasing protein stability: using an empirical approach of exploring naturally occurring variations among a family of homologous proteins, FIGURE 5: Superposition of the structures of WF59 (white bonds) and wild-type (yellow bonds) FKBP12 shown in stereo. Drawn with MolScript (Kraulis, 1991) and Raster3D (Merrit and Murphy, 1994): (a) R-helix and residues surrounding mutation site; (b) loop4 region (residues 50-54) showing the hydrogen bonds between Glu60 (R-helix) and the backbone amide NH groups of residues 51 and 53. single stabilizing mutations have been combined to create a superstable multiple mutant for the proteins: barnase (Serrano et al, 1993); GroEL apical domain (Wang et al, 2000); barstar (Schreiber et al, 1994); and p53 (Nikolova et al, 1998).…”

Section: Increased Stability Of Mutants Suggests a Tradeoff Betweenmentioning

confidence: 99%

Energetic and Structural Analysis of the Role of Tryptophan 59 in FKBP12

Fulton¹,

Jackson

Buckle

2003

Biochemistry

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Tryptophan 59 forms the seat of the hydrophobic ligand-binding site in the small immunophilin FKBP12. Mutating this residue to phenylalanine or leucine stabilizes the protein by 2.72 and 2.35 kcal mol -1 , respectively. Here we report the stability data and 1.7 Å resolution crystal structures of both mutant proteins, complexed with the immunosuppressant rapamycin. Both structures show a relatively large response to mutation involving a helical bulge at the mutation site and the loss of a hydrogen bond that anchors a nearby loop. The increased stability of the mutants is probably due to a combination of improved packing and an entropic gain at the mutation site. The structures are almost identical to that of wild-type FKBP12.6, an isoform of FKBP12 that differs by 18 residues, including Trp59, in its sequence. Therefore, the structural difference between the two isoforms can be attributed almost entirely to the identity of residue 59. It is likely that in FKBP12-ligand complexes Trp59 provides added binding energy at the active site at the expense of protein stability, a characteristic common to other proteins. FKBP12 associates with the ryanodine receptor in skeletal muscle (RyR1), while FKBP12.6 selectively binds the ryanodine receptor in cardiac muscle (RyR2). The structural response to mutation suggests that residue 59 contributes to the specificity of binding between FKBP12 isoforms and ryanodine receptors.

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“…sheettracer then employs a multi-step process to build pseudo-C a traces in each identified sheet. These steps are first illustrated using as an example one b-sheet of the apical domain of the molecular chaperonin GroEL, also known as the minichaperone 17 (PDB code 1fy9).…”

Section: Resultsmentioning

confidence: 99%

A Structural-informatics Approach for Tracing β-Sheets: Building Pseudo-Cα Traces for β-Strands in Intermediate-resolution Density Maps

Kong

Zhang

Baker

et al. 2004

Journal of Molecular Biology

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We report the development of two computational methods to assist density map interpretation at intermediate resolutions: sheettracer for building pseudo-C(alpha) models of beta-sheets, and a deconvolution method for enhancing features attributed to major secondary structural elements. Sheettracer is tightly coupled with sheetminer, which was developed to locate sheet densities in intermediate-resolution density maps. The results from sheetminer are used as inputs to sheettracer, which employs a multi-step ad hoc morphological analysis of sheet densities to trace individual strands of beta-sheets. The methods were tested on simulated density maps from 12 protein crystal structures that represent a reasonably complete sampling of sheet morphology. The sheet-tracing results were quantitatively assessed in terms of sensitivity, specificity and rms deviations. Furthermore, sheettracer and the deconvolution method were rigorously tested on experimental maps of the lambda2 protein of reovirus at resolutions of 7.6A and 11.8A. Our results clearly demonstrate the capability of sheettracer in building pseudo-C(alpha) models of beta-sheets in intermediate-resolution density maps and the power of the deconvolution method in enhancing the performance of sheettracer. These computational methods, along with other related ones, should facilitate recognition and analysis of folding motifs from experimental data at intermediate resolutions.

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Stabilization of GroEL minichaperones by core and surface mutations

Cited by 36 publications

References 39 publications

The High Resolution Crystal Structure of a Native Thermostable Serpin Reveals the Complex Mechanism Underpinning the Stressed to Relaxed Transition

The High Resolution Crystal Structure of a Native Thermostable Serpin Reveals the Complex Mechanism Underpinning the Stressed to Relaxed Transition

Energetic and Structural Analysis of the Role of Tryptophan 59 in FKBP12

A Structural-informatics Approach for Tracing β-Sheets: Building Pseudo-Cα Traces for β-Strands in Intermediate-resolution Density Maps

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