Fumihiro Motojima

Ribosomal protein L2 associates with E. coli HtpG and activates its ATPase activity

Although eukaryotic Hsp90 has been studied extensively, the function of its bacterial homologue HtpG remains elusive. Here we report that 50S ribosomal protein L2 was found as an associated protein with His-tagged HtpG from Escherichia coli cultured in minimum medium at 45 °C. L2 specifically activated ATPase activity of HtpG, but other denatured proteins did not. The analysis using domain derivatives of HtpG and L2 showed that C-terminal domain of L2 and the middle to C-terminal domain of HtpG are important for interaction. At physiological salt concentration, L2 was denatured state and was recognized by HtpG as well as other chaperones, DnaK/DnaJ/GrpE and GroEL/GroES. The ATPase of HtpG at increasing concentration of L2 indicated that an L2 molecule bound to a dimer HtpG with apparent K(D) of 0.3 μM at 100mM KCl and 3.3 μM at 200 mM KCl.

Polypeptide in the chaperonin cage partly protrudes out and then folds inside or escapes outside

The current mechanistic model of chaperonin‐assisted protein folding assumes that the substrate protein in the cage, formed by GroEL central cavity capped with GroES, is isolated from outside and exists as a free polypeptide. However, using ATPase‐deficient GroEL mutants that keep GroES bound, we found that, in the rate‐limiting intermediate of a chaperonin reaction, the unfolded polypeptide in the cage partly protrudes through a narrow space near the GroEL/GroES interface. Then, the entire polypeptide is released either into the cage or to the outside medium. The former adopts a native structure very rapidly and the latter undergoes spontaneous folding. Partition of the in‐cage folding and the escape varies among substrate proteins and is affected by hydrophobic interaction between the polypeptide and GroEL cavity wall. The ATPase‐active GroEL with decreased in‐cage folding produced less of a native model substrate protein in Escherichia coli cells. Thus, the polypeptide in the critical GroEL–GroES complex is neither free nor completely confined in the cage, but it is interacting with GroELs apical region, partly protruding to outside.

Rotation of the γ Subunit in F1-ATPase; Evidence That ATP Synthase Is a Rotary Motor Enzyme

ATP-dependent, azide-sensitive rotation of the γ subunit relative to the α3β3 hexagonal ring of ATP synthase was observed with a single molecule imaging system. Thus, ATP synthase is a rotary motor enzyme, the first ever found.

Determination of the Number of Active GroES Subunits in the Fused Heptamer GroES Required for Interactions with GroEL

A double-heptamer ring chaperonin GroEL binds denatured substrate protein, ATP, and GroES to the same heptamer ring and encapsulates substrate into the central cavity underneath GroES where productive folding occurs. GroES is a disk-shaped heptamer, and each subunit has a GroEL-binding loop. The residues of the GroEL subunit responsible for GroES binding largely overlap those involved in substrate binding, and the mechanism by which GroES can replace the substrate when GroES binds to GroEL/substrate complex remains to be clarified. To address this question, we generated single polypeptide GroES by fusing seven subunits with various combinations of active and GroEL binding-defective subunits. Functional tests of the fused GroES variants indicated that four active GroES subunits were required for efficient formation of the stable GroEL/GroES complex and five subunits were required for the productive GroEL/substrate/GroES complex. An increase in the number of defective GroES subunits resulted in a slowing of encapsulation and folding. These results indicate the presence of an intermediate GroEL/substrate/GroES complex in which the substrate and GroES bind to GroEL by sharing seven common binding sites.

Nuclear magnetic resonance approaches for characterizing interactions between the bacterial chaperonin GroEL and unstructured proteins.

GroEL-protein interactions were characterized by stable isotope-assisted nuclear magnetic resonance (NMR) spectroscopy using chemically denatured bovine rhodanese and an intrinsically disordered protein, α-synuclein, as model ligands. NMR data indicated that proteins tethered to GroEL remain largely unfolded and highly mobile, enabling identification of the interaction hot spots displayed on intrinsically disordered proteins.

Revisiting the contribution of negative charges on the chaperonin cage wall to the acceleration of protein folding

Chaperonin GroEL mediates the folding of protein encapsulated in a GroES-sealed cavity (cage). Recently, a critical role of negative charge clusters on the cage wall in folding acceleration was proposed based on experiments using GroEL single-ring (SR) mutants SR1 and SRKKK2 [Tang YC, et al. (2006) Cell 125:903–914; Chakraborty K, et al. (2010) Cell 142:112–122]. Here, we revisited these experiments and discovered several inconsistencies. (i) SR1 was assumed to bind to GroES stably and to mediate single-round folding in the cage. However, we show that SR1 repeats multiple turnovers of GroES release/binding coupled with ATP hydrolysis. (ii) Although the slow folding observed for a double-mutant of maltose binding protein (DMMBP) by SRKKK2 was attributed to mutations that neutralize negative charges on the cage wall, we found that the majority of DMMBP escape from SRKKK2 and undergo spontaneous folding in the bulk medium. (iii) An osmolyte, trimethylamine N-oxide, was reported to accelerate SRKKK2-mediated folding of DMMBP by mimicking the effect of cage-wall negative charges of WT GroEL and ordering the water structure to promote protein compaction. However, we demonstrate that in-cage folding by SRKKK2 is unaffected by trimethylamine N-oxide. (iv) Although it was reported that SRKKK2 lost the ability to assist the folding of ribulose-1,5-bisphosphate carboxylase/oxygenase, we found that SRKKK2 retains this ability. Our results argue against the role of the negative charges on the cage wall of GroEL in protein folding. Thus, in chaperonin studies, folding kinetics need to be determined from the fraction of the real in-cage folding.

Preliminary X-ray crystallographic studies of photosynthetic reaction center from a thermophilic sulfur bacterium, Chromatium tepidum.

A membrane protein complex, photosynthetic reaction center purified from the thermophilic purple sulfur bacterium, Chromatium tepidum has been crystallized from a phosphate butter containing a detergent, n‐octyl‐β‐D‐glucopyranoside and a precipitant, polyethylene glycol 4000. The crystals diffracted X‐rays beyond 3Å resolution with synchrotron radiation and are suitable for high‐resolution X‐ray crystallographic studies. The crystals belong to the orthorhombic space group P212121 with unit‐cell dimensions of a = 136Å, b = 197Å, and c = 82Å. Assuming that they contain one reaction center complex in the asymmetric unit, V M was calculated to be 4.3 Å3/Da, which agrees with the values obtained in the membrane protein complexes.

How do chaperonins fold protein

Protein folding is a biological process that is essential for the proper functioning of proteins in all living organisms. In cells, many proteins require the assistance of molecular chaperones for their folding. Chaperonins belong to a class of molecular chaperones that have been extensively studied. However, the mechanism by which a chaperonin mediates the folding of proteins is still controversial. Denatured proteins are folded in the closed chaperonin cage, leading to the assumption that denatured proteins are completely encapsulated inside the chaperonin cage. In contrast to the assumption, we recently found that denatured protein interacts with hydrophobic residues at the subunit interfaces of the chaperonin, and partially protrude out of the cage. In this review, we will explain our recent results and introduce our model for the mechanism by which chaperonins accelerate protein folding, in view of recent findings.

Productive folding of a tethered protein in the chaperonin GroEL-GroES cage.

Many proteins in bacterial cells fold in the chaperonin cage made of the central cavity of GroEL capped by GroES. Recent studies indicate that the polypeptide in the cage spends the most time as a state tethered dynamically to the GroEL/GroES interface region, in which folding occurs in the polypeptide segments away from the tethered site (F. Motojima & M. Yoshida, EMBO J. (2010) 29, 4008-4019). In support of this, we show here that a polypeptide in the cage tethered covalently to an appropriate site in the GroEL/GroES interface region can fold to a near-native structure.

Physicochemical Properties of the Mammalian Molecular Chaperone HSP60

The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football- and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle.