Fernando Moro

The TIM17·23 preprotein translocase of mitochondria: composition and function in protein transport into the matrix

We have analysed the structural organization of the TIM17·23 complex, the preprotein translocase of the mitochondrial inner membrane specific for protein targeting to the matrix. The components Tim17, Tim23 and Tim44 are present in this complex in equimolar amounts. A sub‐complex containing Tim23 and Tim44 but no Tim17, or a sub‐complex containing Tim23 and Tim17 but no Tim44 was not detected. Tim44 is peripherally associated at the matrix side. Tim44 forms dimers which recruit two molecules of mt‐Hsp70 to the sites of protein import. A sequential, hand‐over‐hand mode of interaction of these two mt‐Hsp70·Tim44 complexes with a translocating polypeptide chain is proposed.

The structure of CCT-Hsc70 NBD suggests a mechanism for Hsp70 delivery of substrates to the chaperonin

Chaperones, a group of proteins that assist the folding of other proteins, seem to work in a coordinated manner. Two major chaperone families are heat-shock protein families Hsp60 and Hsp70. Here we show for the first time the formation of a stable complex between chaperonin-containing TCP-1 (CCT) and Hsc70, two eukaryotic representatives of these chaperone families. This interaction takes place between the apical domain of the CCTβ subunit and the nucleotide binding domain of Hsc70, and may serve to deliver the unfolded substrate from Hsc70 to the substrate binding region of CCT. We also show that a similar interaction does not occur between their prokaryotic counterparts GroEL and DnaK, suggesting that in eukarya the two types of chaperones have evolved to a concerted action that makes the folding task more efficient.

Interdomain interaction through helices A and B of DnaK peptide binding domain.

In order to better define the structural elements involved in allosteric signalling, wild‐type DnaK and three deletion mutants of the peptide binding domain have been characterized by biophysical (steady‐state and time‐resolved fluorescence) and biochemical methods. In the presence of ATP the chemical environment of the single tryptophan residue of DnaK, located in the ATPase domain, becomes less polar, as seen by a blue shift of the emission maximum and a shortening of the fluorescence lifetime, and its accessibility to polar quenchers is drastically reduced. These nucleotide‐dependent modifications are also observed for the deletion mutant DnaK1‐537, but not for DnaK1‐507 or DnaK1‐385, and thus rely on the presence of residues 507–537 (helices A and the N‐terminal half of B) of the peptide binding domain. These data indicate that αA and half αB contribute to the allosteric communication of DnaK. In the presence of ATP, they promote a conformational change that displaces a residue(s) of the peptide binding domain towards a region of the ATPase domain where the tryptophan residue (W102) is located. A putative role for these helical segments as regulators of the position of the lid is discussed.

DnaK-mediated association of ClpB to protein aggregates. A bichaperone network at the aggregate surface.

MINT‐7258974: DnaK (uniprotkb:P0A6Y8), ClpB (uniprotkb:P63284), DnaJ (uniprotkb:P08622) and G6PDH (uniprotkb:P0AC53) physically interact (MI:0914) by cosedimentation (MI:0027)

Role of DnaJ G/F-rich Domain in Conformational Recognition and Binding of Protein Substrates

DnaJ from Escherichia coli is a Type I Hsp40 that functions as a cochaperone of DnaK (Hsp70), stimulating its ATPase activity and delivering protein substrates. How DnaJ binds protein substrates is still poorly understood. Here we have studied the role of DnaJ G/F-rich domain in binding of several substrates with different conformational properties (folded, partially (un)folded and unfolded). Using partial proteolysis we find that RepE, a folded substrate, contacts a wide DnaJ area that involves part of the G/F-rich region and Zn-binding domain. Deletion of G/F-rich region hampers binding of native RepE and reduced the affinity for partially (un)folded substrates. However, binding of completely unfolded substrates is independent on the G/F-rich region. These data indicate that DnaJ distinguishes the substrate conformation and is able to adapt the use of the G/F-rich region to form stable substrate complexes.

Ionic contacts at DnaK substrate binding domain involved in the allosteric regulation of lid dynamics.

To gain further insight into the interactions involved in the allosteric transition of DnaK we have characterized wild-type (wt) protein and three mutants in which ionic interactions at the interface between the two subdomains of the substrate binding domain, and within the lid subdomain have been disrupted. Our data show that ionic contacts, most likely forming an electrically charged network, between the N-terminal region of helix B and an inner loop of the β-sandwich are involved in maintaining the position of the lid relative to the β-subdomain in the ADP state but not in the ATP state of the protein. Disruption of the ionic interactions between the C-terminal region of helix B and the outer loops of the β-sandwich, known as the latch, does not have the same conformational consequences but results equally in an inactive protein. This indicates that a variety of mechanisms can inactivate this complex allosteric machine. Our results identify the ionic contacts at the subdomain and interdomain interfaces that are part of the hinge region involved in the ATP-induced allosteric displacement of the lid away from the peptide binding site. These interactions also stabilize peptide-Hsp70 complexes at physiological (37 °C) and stress (42 °C) temperatures, a requirement for productive substrate (re)folding.

Fluorescence quenching at interfaces and the permeation of acrylamide and iodide across phospholipid bilayers

Studies of fluorescence quenching in membrane proteins are complicated by the fact that the barrier effect of the bilayer towards the quenchers is not known with precision. Our studies show that (a) both acrylamide and iodide can permeate the membrane at comparable rates, (b) when quenchers are added externally to a vesicle suspension, the apparent Stern‐Volmer quenching constants for the same fluorophores are lower in the inner than in the outer aqueous compartments, and (c) at least some non‐polar fluorophores embedded in the bilayer are quenched by iodide, but not by acrylamide.

DnaJ Recruits DnaK to Protein Aggregates

Thermal stress might lead to protein aggregation in the cell. Reactivation of protein aggregates depends on Hsp100 and Hsp70 chaperones. We focus in this study on the ability of DnaK, the bacterial representative of the Hsp70 family, to interact with different aggregated model substrates. Our data indicate that DnaK binding to large protein aggregates is mediated by DnaJ, and therefore it depends on its affinity for the cochaperone. Mutations in the structural region of DnaK known as the “latch” decrease the affinity of the chaperone for DnaJ, resulting in a defective activity as protein aggregate-removing agent. As expected, the chaperone activity is recovered when DnaJ concentration is raised to overcome the lower affinity of the mutant for the cochaperone, suggesting that a minimum number of aggregate-bound DnaK molecules is necessary for its efficient reactivation. Our results provide the first experimental evidence of DnaJ-mediated recruiting of ATP-DnaK molecules to the aggregate surface.

The allosteric transition in DnaK probed by infrared difference spectroscopy. Concerted ATP-induced rearrangement of the substrate binding domain

The biological activity of DnaK, the bacterial representative of the Hsp70 protein family, is regulated by the allosteric interaction between its nucleotide and peptide substrate binding domains. Despite the importance of the nucleotide‐induced cycling of DnaK between substrate‐accepting and releasing states, the heterotropic allosteric mechanism remains as yet undefined. To further characterize this mechanism, the nucleotide‐induced absorbance changes in the vibrational spectrum of wild‐type DnaK was characterized. To assign the conformation sensitive absorption bands, two deletion mutants (one lacking the C‐terminal α‐helical subdomain and another comprising only the N‐terminal ATPase domain), and a single‐point DnaK mutant (T199A) with strongly reduced ATPase activity, were investigated by time‐resolved infrared difference spectroscopy combined with the use of caged‐nucleotides. The results indicate that (1) ATP, but not ADP, binding promotes a conformational change in both subdomains of the peptide binding domain that can be individually resolved; (2) these conformational changes are kinetically coupled, most likely to ensure a decrease in the affinity of DnaK for peptide substrates and a concomitant displacement of the lid away from the peptide binding site that would promote efficient diffusion of the released peptide to the medium; and (3) the α‐helical subdomain contributes to stabilize the interdomain interface against the thermal challenge and allows bidirectional transmission of the allosteric signal between the ATPase and substrate binding domains at stress temperatures (42°C).

A quantitative analysis of the effect of nucleotides and the M domain on the association equilibrium of ClpB.

ClpB is a hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stress-denatured proteins. The hexamer is a highly dynamic complex, able to reshuffle subunits. To further characterize the biological implications of the ClpB oligomerization state, the association equilibrium of the wild-type (wt) protein and of two deletion mutants, which lack part or the whole M domain, was quantitatively analyzed under different experimental conditions, using several biophysical [analytical ultracentrifugation, composition-gradient (CG) static light scattering, and circular dichroism] and biochemical (ATPase and chaperone activity) methods. We have found that (i) ClpB self-associates from monomers to form hexamers and higher-order oligomers that have been tentatively assigned to dodecamers, (ii) oligomer dissociation is not accompanied by modifications of the protein secondary structure, (iii) the M domain is engaged in intersubunit interactions that stabilize the protein hexamer, and (iv) the nucleotide-induced rearrangement of ClpB affects the protein oligomeric core, in addition to the proposed radial extension of the M domain. The difference in the stability of the ATP- and ADP-bound states [ΔΔG(ATP-ADP) = -10 kJ/mol] might explain how nucleotide exchange promotes the conformational change of the protein particle that drives its functional cycle.