The Trimeric Major Capsid Protein of Mavirus is stabilized by its Interlocked N-termini Enabling Core Flexibility for Capsid Assembly

Icosahedral viral capsids assemble with high fidelity from a large number of identical buildings blocks. The mechanisms that enable individual capsid proteins to form stable oligomeric units (capsomers) while affording structural adaptability required for further assembly into capsids are mostly unknown. Understanding these mechanisms requires knowledge of the capsomers’ dynamics, especially for viruses where no additional helper proteins are needed during capsid assembly like for the Mavirus virophage that despite its complexity (triangulation number T = 27) can assemble from its major capsid protein (MCP) alone. This protein forms the basic building block of the capsid namely a trimer (MCP$_{3}$) of double-jelly roll protomers with highly intertwined N-terminal arms of each protomer wrapping around the other two at the base of the capsomer, secured by a clasp that is formed by part of the C-terminus. Probing the dynamics of the capsomer with HDX mass spectrometry we observed differences in conformational flexibility between functional elements of the MCP trimer. While the N-terminal arm and clasp regions show above average deuterium incorporation, the two jelly-roll units in each protomer also differ in their structural plasticity, which might be needed for efficient assembly. Assessing the role of the N-terminal arm in maintaining capsomer stability showed that its detachment is required for capsomer dissociation, constituting a barrier towards capsomer monomerisation. Surprisingly, capsomer dissociation was irreversible since it was followed by a global structural rearrangement of the protomers as indicated by computational studies showing a rearrangement of the N-terminus blocking part of the capsomer forming interface

crystal and solution structures of the multidomain cochaperone DnaJ

Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin- labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.1\. Auflag

Folding Properties of the Nucleotide Exchange Factor GrpE from Thermus thermophilus: GrpE is a Thermosensor that Mediates Heat Shock Response

Hsp70 proteins like DnaK bind unfolded polypeptides in a nucleotide-dependent manner. The switch from high-affinity ADP-state to low- affinity ATP-state with concomitant substrate release is accelerated significantly by GrpE proteins. GrpE thus fulfils an important role in regulation of the chaperone cycle. Here, we analysed the thermal stability of GrpE from Thermus thermophilus using differential scanning calorimetry and CD-spectroscopy. The protein exhibits unusual unfolding characteristics with two observable thermal transitions. The first transition is CD-spectroscopically silent with a transition midpoint at 90 °C. The second transition, mainly constituting the CD-signal, ranges between 100 and 105 °C depending on the GrpETth concentration, according to the model [formula]. Using a C-terminally truncated version of GrpETth it was possible to assign the second thermal transition to the dimerisation of GrpETth, while the first transition represents the completely reversible unfolding of the globular C-terminal domain. The unfolding of this domain is accompanied by a distinct decrease in nucleotide exchange rates and impaired binding to DnaKTth. Under heat shock conditions, the DnaK·ADP·protein-substrate complex is thus stabilised by a reversibly inactivated GrpE-protein that refolds under permissive conditions. In combination with studies on GrpE from Escherichia coli presented recently by Christen and co-workers, it thus appears that the general role of GrpE is to function as a thermosensor that modulates nucleotide exchange rates in a temperature-dependent manner to prevent substrate dissociation at non-permissive conditions

NSC249308

The enzyme form of the calcium adenosinetriphosphatase of sarcoplasmic reticulum (CaATPase) that is stable in the presence of calcium, cE.Ca2, has a binding site for the catalytic Mg2+ ion with a dissociation constant of 0.94 +/− 0.15 mM at 25 degrees C, pH 7.0, and 100 mM KCl. This is approximately 10 times smaller than that reported for the free enzyme, E, (8.8 mM) under similar conditions [Punzengruber, C., Prager, R., Kolassa, N., Winkler, F., & Suko, J. (1978) Eur. J. Biochem. 92, 349−359]. This difference shows that the sites for the catalytic and the transported ions interact in the absence of ATP. The addition of ATP and EDTA to enzyme that had been incubated with Ca2+ and Mg2+ resulted in the formation of 61% phosphoenzyme. The addition of unlabeled ATP and Mg2+ to enzyme that had been incubated with 3.5 microM free Ca2+ and labeled ATP gave 39% labeled phosphoenzyme. This shows that the binding of ATP and Mg2+ to cE.Ca2 follows a random mechanism. The rate constants for dissociation of ATP and Mg2+ from cE.Ca2.ATP.Mg are different: kdiss(ATP) = 120 s−1 and kdiss(Mg2+) = 60 s−1. This shows that Mg2+ and ATP can bind and dissociate independently; they do not have to associate or dissociate from cE as a Mg.ATP complex. Calcium−free enzyme binds metal−free ATP at the active site with a dissociation constant of 44 +/− 4 microM, kdiss = 130 +/− 7 s−1, and a calculated association rate constant of 3 x 10(6) M−1 s−1. Calcium−free enzyme that was incubated with [gamma−32P]ATP gave 38% labeled phosphoenzyme when chased with unlabeled ATP, Mg2+, and Ca2+. An increase of the Mg2+ concentration did not increase the amount of E32P formed. This shows that the binding of Mg2+ and ATP to free E also follows a random mechanism. The Mg2+ ion is not buried under ATP, and ATP is not under a Mg2+ ion. Incubation of free E with Mg2+ and ATP causes a conformational change that activates the enzyme for phosphorylation and decreases the rate constant for the dissociation of ATP from kdiss = 120 s−1 to kdiss = 47 s−

Coupling and dynamics of subunits in the hexameric AAA+ chaperone ClpB

The bacterial AAA+ protein ClpB and its eukaryotic homologue Hsp104 ensure thermotolerance of their respective organisms by reactivating aggregated proteins in cooperation with the Hsp70/Hsp40 chaperone system. Like many members of the AAA+ superfamily, the ClpB protomers form ringlike homohexameric complexes. The mechanical energy necessary to disentangle protein aggregates is provided by ATP hydrolysis at the two nucleotide−binding domains of each monomer. Previous studies on ClpB and Hsp104 show a complex interplay of domains and subunits resulting in homotypic and heterotypic cooperativity. Using mutations in the Walker A and Walker B nucleotide−binding motifs in combination with mixing experiments we investigated the degree of inter−subunit coupling with respect to different aspects of the ClpB working cycle. We find that subunits are tightly coupled with regard to ATPase and chaperone activity, but no coupling can be observed for ADP binding. Comparison of the data with statistical calculations suggests that for double Walker mutants, approximately two in six subunits are sufficient to abolish chaperone and ATPase activity completely. In further experiments, we determined the dynamics of subunit reshuffling. Our results show that ClpB forms a very dynamic complex, reshuffling subunits on a timescale comparable to steady−state ATP hydrolysis. We propose that this could be a protection mechanism to prevent very stable aggregates from becoming suicide inhibitors for ClpB

Modulation of the ATPase cycle of BiP by peptides and proteins

BiP, the Hsp70 homologue of the endoplasmic reticulum, interacts with its non-native substrate proteins in an ATP-dependent manner. This interaction is coupled to the ATPase cycle of the chaperone. Binding of short, synthetic peptides stimulate the ATPase activity of BiP. In previous work, we showed that a stably unfolded antibody domain forms a binary complex with BiP. In this study we made use of this complex to analyse the effect of substrate proteins on the ATPase cycle of BiP. Kinetic constants of the partial reactions of the ATPase cycle were determined without substrate, in the presence of a short binding peptide and in the presence of the antibody domain. We show that, in contrast to smaller peptides, the non-native protein domain decelerates the rate limiting hydrolysis step of the ATPase cycle