Temperature Dependence of Backbone Dynamics in Loops of Human Mitochondrial Heat Shock Protein 10

Landry, Samuel J.; Nk, Steede; Maskos, Karol

doi:10.1021/bi971141p

Cited by 39 publications

(40 citation statements)

References 77 publications

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“…This is corroborated by nuclear magnetic resonance measurements, which show turns in the base loops of GroES and Gp31 as part of a ␤-hairpin structure when bound to GroEL (22). X-ray crystallographic and nuclear magnetic resonance data indicate that the base loop of human cpn10 also preferentially samples a hairpin conformation (21). However, there is conformational variability in the base loop sequences surrounding the ␤-turn in all these structures.…”

Section: Dynamic Light Scattering (Dls)mentioning

confidence: 61%

Mycobacterium tuberculosisChaperonin 10 Heptamers Self-Associate through Their Biologically Active Loops

et al. 2003

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The crystal structure of Mycobacterium tuberculosis chaperonin 10 (cpn10 Mt ) has been determined to a resolution of 2.8 Å. Two dome-shaped cpn10 Mt heptamers complex through loops at their bases to form a tetradecamer with 72 symmetry and a spherical cage-like structure. The hollow interior enclosed by the tetradecamer is lined with hydrophilic residues and has dimensions of 30 Å perpendicular to and 60 Å along the sevenfold axis. Tetradecameric cpn10 Mt has also been observed in solution by dynamic light scattering. Through its base loop sequence cpn10 Mt is known to be the agent in the bacterium responsible for bone resorption and for the contribution towards its strong T-cell immunogenicity. Superimposition of the cpn10 Mt sequences 26 to 32 and 66 to 72 and E. coli GroES 25 to 31 associated with bone resorption activity shows them to have similar conformations and structural features, suggesting that there may be a common receptor for the bone resorption sequences. The base loops of cpn10s in general also attach to the corresponding chaperonin 60 (cpn60) to enclose unfolded protein and to facilitate its correct folding in vivo. Electron density corresponding to a partially disordered protein subunit appears encapsulated within the interior dome cavity of each heptamer. This suggests that the binding of substrates to cpn10 is possible in the absence of cpn60.The 10-kDa chaperonin of Mycobacterium tuberculosis (cpn10 Mt ) (3) is strongly implicated as an important virulence factor during infection (10, 28). Investigation of the structural and functional characteristics of the protein is therefore an imperative in the search for routes to combat this persistent human pathogen. While the fundamental role of chaperonins as facilitators of protein folding within the cell is well established (13, 41), the secretion of cpn10 Mt into the extracellular space (1, 4) and its pathogenic properties are poorly understood. cpn10 (GroES) acts in concert with the 60-kDa chaperonin (cpn60), GroEL, ATP, and a repertoire of other molecular chaperones to catalyze protein folding (39, 47). The genome for M. tuberculosis contains the two GroEL homologues cpn60.1 Mt and cpn60.2 Mt (17) that presumably function in concert with cpn10 Mt to enable protein folding in the mycobacterium to occur and to contribute to the stress response of the organism that resides in the hostile interior of the macrophage. Mitochondrial cpn60 is also located on the surface of cells, suggesting that it could also serve as a receptor for extracellular cpn10 (42).cpn10 Mt has a potent bone-resorbing activity by promoting the recruitment of osteoclasts and inhibiting the growth of osteoblasts (29). This is believed to be a central feature of the pathology of spinal tuberculosis, one of the more severe diseases associated with M. tuberculosis infection, and represents a cell signaling function of the secreted protein.Healthy bone is maintained by a dynamic equilibrium between the bone matrix-forming osteoblast cells and the bone-resorbing osteoclast cells (32...

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Section: Dynamic Light Scattering (Dls)mentioning

confidence: 61%

Mycobacterium tuberculosisChaperonin 10 Heptamers Self-Associate through Their Biologically Active Loops

et al. 2003

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“…Although the roof ␤-hairpin is much smaller than the mobile loop, five residues in the roof ␤-hairpin have higher than average crystallographic B-factors (Fig. 2), and the corresponding loop in human Hsp10 is highly flexible (22). The roof ␤-hairpin also was able to present a continuous epitope to antibodies.…”

Section: Discussionmentioning

confidence: 98%

Structural Basis for Helper T-cell and Antibody Epitope Immunodominance in Bacteriophage T4 Hsp10

Dai

Carmicle

Steede

et al. 2002

Journal of Biological Chemistry

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Antigen three-dimensional structure potentially limits the access of endoproteolytic processing enzymes to cleavage sites and of class II major histocompatibility antigen-presenting proteins to helper T-cell epitopes. Helper T-cell epitopes in bacteriophage T4 Hsp10 have been mapped by restimulation of splenocytes from CBA/J and C57BL/6J mice immunized in conjunction with mutant (R192G) heat-labile enterotoxin from Escherichia coli. Promiscuously immunogenic sequences were associated with unstable loops in the three-dimensional structure of T4 Hsp10. The immunodominant sequence lies on the N-terminal flank of the 22-residue mobile loop, which is sensitive to proteolysis in divergent Hsp10s. Several mobile loop deletions that inhibited proteolysis in vitro caused global changes in the helper T-cell epitope map. A mobile loop deletion that strongly stabilized the protein dramatically reduced the immunogenicity of the flanking immunodominant helper T-cell epitope, although the protein retained good overall immunogenicity. Antisera against the mobile loop deletion variants exhibited increased crossreactivity, most especially the antisera against the strongly stabilized variant. The results support the hypothesis that unstable loops promote the presentation of flanking epitopes and suggest that loop deletion could be a general strategy to increase the breadth and strength of an immune response.

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“…Hsp10 was purified as described by Landry et al (31). All proteins were stored at Ϫ80°C in a buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, and 15% (v/v) glycerol.…”

Section: Methodsmentioning

confidence: 99%

“…Indeed, it is known that mutations at this position, P33S or P33H, reduce the affinity of yeast Hsp10 for GroEL (33,34). Most likely, substitution at this site results in increased conformational dynamics at position 33, which in turn increases the entropic cost of binding to GroEL (31,54). Finally, the nature of the residue at position 21 in GroES and the corresponding position 26 in Hsp10 is important in determining ␤-sheet propensity.…”

Section: A Groes Mutant Protein Carrying Three Amino Acid Substitutiomentioning

confidence: 99%

The Importance of a Mobile Loop in Regulating Chaperonin/ Co-chaperonin Interaction

Richardson¹,

Schwager²,

Landry³

et al. 2001

Journal of Biological Chemistry

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Chaperonins are universally conserved proteins that nonspecifically facilitate the folding of a wide spectrum of proteins. While bacterial GroEL is functionally promiscuous with various co-chaperonin partners, its human homologue, Hsp60 functions specifically with its co-chaperonin partner, Hsp10, and not with other cochaperonins, such as the bacterial GroES or bacteriophage T4-encoded Gp31. Co-chaperonin interaction with chaperonin is mediated by the co-chaperonin mobile loop that folds into a ␤-hairpin conformation upon binding to the chaperonin. A delicate balance of flexibility and conformational preferences of the mobile loop determines co-chaperonin affinity for chaperonin. Here, we show that the ability of Hsp10, but not GroES, to interact specifically with Hsp60 lies within the mobile loop sequence. Using mutational analysis, we show that three substitutions in the GroES mobile loop are necessary and sufficient to acquire Hsp10-like specificity. Two of these substitutions are predicted to preorganize the ␤-hairpin turn and one to increase the hydrophobicity of the GroEL-binding site. Together, they result in a GroES that binds chaperonins with higher affinity. It seems likely that the single ring mitochondrial Hsp60 exhibits intrinsically lower affinity for the co-chaperonin that can be compensated for by a higher affinity mobile loop.

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Temperature Dependence of Backbone Dynamics in Loops of Human Mitochondrial Heat Shock Protein 10

Cited by 39 publications

References 77 publications

Mycobacterium tuberculosisChaperonin 10 Heptamers Self-Associate through Their Biologically Active Loops

Mycobacterium tuberculosisChaperonin 10 Heptamers Self-Associate through Their Biologically Active Loops

Structural Basis for Helper T-cell and Antibody Epitope Immunodominance in Bacteriophage T4 Hsp10

The Importance of a Mobile Loop in Regulating Chaperonin/ Co-chaperonin Interaction

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