Chemical cross‐linking of the chloroplast localized small heat‐shock protein, Hsp21, and the model substrate citrate synthase

Abstract

The molecular mechanism whereby the small heat-shock protein (sHsp) chaperones interact with and prevent aggregation of other proteins is not fully understood. We have characterized the sHsp-substrate protein interaction at normal and increased temperatures utilizing a model substrate protein, citrate synthase (CS), widely used in chaperone assays, and a dodecameric plant sHsp, Hsp21, by chemical cross-linking with 3,39-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) and mass spectrometric peptide mapping. In the absence of CS, the cross-linker captured Hsp21 in dodecameric form, even at increased temperature (47°C). In the presence of equimolar amounts of CS, no Hsp21 dodecamer was captured, indicating a substrate-induced Hsp21 dodecamer dissociation by equimolar amounts of CS. Cross-linked Hsp21-Hsp21 dipeptides indicated an exposure of the Hsp21 C-terminal tails and substrate-binding sites normally covered by the C terminus. Cross-linked Hsp21-CS dipeptides mapped to several sites on the surface of the CS dimer, indicating that there are numerous weak and short-lived interactions between Hsp21 and CS, even at normal temperatures. The N-terminal arms especially interacted with a motif in the CS dimer, which is absent in thermostable forms of CS. The cross-linking data suggest that the presence of substrate rather than temperature influences the conformation of Hsp21.Keywords: chemical cross-linking; mass spectrometric peptide mapping; small heat-shock protein; protein-protein interactions; citrate synthase Small heat-shock proteins (sHsps) exist in essentially all organisms and are especially abundant in plants (Waters et al. 1996;Haslbeck et al. 2005). The sHsps have a particularly important role during heat stress, where they protect other proteins from aggregation (Horwitz 1992; Narberhaus 2002) and prevent irreversible accumulation of large aggregates within the cell (Sun and MacRae 2005). By maintaining unfolded proteins in a folding-competent state, sHsps work with the ATP-dependent Hsp70 systems, which can then refold the proteins when the stress conditions return to normal (van Montfort et al. 2001b;Haslbeck 2002;Stromer et al. 2003;Haslbeck et al. 2004).The sHsp monomers range from 12 to 43 kDa and assemble into large multimers (van Montfort et al. 2001b;Haslbeck et al. 2005). The three-dimensional structures have been determined for the plant Hsp16.9 from wheat Reprint requests to: Emma Å hrman, Department of Biochemistry, Lund University, P.O.Box 124, S-22100, Lund, Sweden; e-mail: emma. ahrman@biochemistry.lu.se; fax: 46-46-222-4534.Abbreviations: DTSSP, 3,39-Dithiobis(sulfosuccinimidylpropionate); DTT, dithiothreitol; MDH, malate dehydrogenase; CS, citrate synthase; nanoESI-MS, nanoelectrospray-ionization mass spectrometry.Article published online ahead of print. Article and publication date are at

Substrate binding site flexibility of the small heat shock protein molecular chaperones

“…The combination of multiple N-terminal binding sites appears to form the highest affinity interaction with substrate compared with other regions of the sHSP. Previous studies have suggested there are both high and low affinity substrate binding sites on sHSPs (25,29). Our results complement this idea by demonstrating that sHSPs can bind to substrate through a plastic interaction surface.…”

“…However, these data do not distinguish between disruption of substrate interaction sites on the N-terminal arm, versus perturbation of some other sHSP property, such as oligomer integrity, which then indirectly impacts chaperone activity. Other data suggest there are additional substrate binding sites on the ␣-crystallin domain, particularly in certain regions involved in oligomer contacts (10,14,24,25).…”

Characterization of rice small heat shock proteins targeted to different cellular organelles

“…Hsp21 from Arabidopsis was seen to exist as a dodecamer. Numerous substrate-binding sites and intra-oligomer interaction sites were mapped on to its N-terminal arm (Ahrman et al 2007). A structure model of Hsp21, from single-particle electron microscopy studies showed that it too existed as a doubledisc dodecamer, albeit with a relative rotation between the two discs, when compared with the wheat Hsp16.9 dodecamer (Lambert et al 2011).…”

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry

“…Recombinantly expressed Hsp21 from Arabidopsis thaliana (UniProtKB ID P31170) was obtained as previously described (Ahrman et al 2007b). MDH (UniProtKB ID P00346) from Sus scrofa (pig) was purchased from Roche (Basel, Switzerland).…”