Kelly M. Knee

Crystal structures of a group II chaperonin reveal the open and closed states associated with the protein folding cycle

Chaperonins are large protein complexes consisting of two stacked multisubunit rings, which open and close in an ATP-dependent manner to create a protected environment for protein folding. Here, we describe the first crystal structure of a group II chaperonin in an open conformation. We have obtained structures of the archaeal chaperonin from Methanococcus maripaludis in both a peptide acceptor (open) state and a protein folding (closed) state. In contrast with group I chaperonins, in which the equatorial domains share a similar conformation between the open and closed states and the largest motions occurs at the intermediate and apical domains, the three domains of the archaeal chaperonin subunit reorient as a single rigid body. The large rotation observed from the open state to the closed state results in a 65% decrease of the folding chamber volume and creates a highly hydrophilic surface inside the cage. These results suggest a completely distinct closing mechanism in the group II chaperonins as compared with the group I chaperonins.

Formation of amyloid fibrils in vitro from partially unfolded intermediates of human γC-crystallin.

PURPOSE Mature-onset cataract results from the formation of light-scattering aggregates of lens crystallins. Although oxidative or mutational damage may be a prerequisite, little is known of the initiation or nucleation of these aggregated states. In mice carrying mutations in gamma-crystallin genes, a truncated form of gamma-crystallin formed intranuclear filamentous inclusions within lens fiber cells. Previous studies have shown that bovine crystallins and human gammaD-crystallin form amyloid fibrils under denaturing conditions in vitro. The amyloid fibril formation of human gammaC-crystallin (HgammaC-Crys) induced by low pH, together with characterization of a partially unfolded intermediate in the process were investigated. METHODS HgammaC-Crys was expressed and purified from Escherichia coli. Partially unfolded intermediates were detected by tryptophan fluorescence spectroscopy and UV resonance Raman spectroscopy. The aggregation into amyloid fibrils was monitored by solution turbidity and fluorescence assay. The morphology of aggregates was characterized using transmission electron microscopy (TEM). Secondary structure of the peptides in their fibrillar state was characterized using Fourier transform infrared spectroscopy (FTIR). RESULTS The structure of HgammaC-Crys was perturbed at low pH. Partially unfolded intermediates were detected when solution pH was lowered to pH 3. At pH 3, HgammaC-Crys aggregated into amyloid fibrils. The kinetics and extent of the reaction was dependent on protein concentration, pH, and temperature. TEM images of aggregates revealed aggregation stages from short to long fibrils and from long fibrils to light-scattering fibril networks. FTIR spectroscopy confirmed the cross-beta character of the secondary structure of these fibrils. CONCLUSIONS HgammaC-Crys formed amyloid fibrils on incubation at low pH via a partially unfolded intermediate. This process could contribute to the early stages of the formation of light-scattering species in the eye lens.

Spectroscopic and Molecular Dynamics Evidence for a Sequential Mechanism for the A-to-B Transition in DNA ☆

The A-to-B form transition has been examined in three DNA duplexes, d(CGCGAATTCGCG)(2), d(CGCGAATTGCGC), and d(CGCAAATTTCGC), using circular dichroism spectroscopy, ultraviolet resonance Raman (UVRR) spectroscopy, and molecular dynamics (MD) simulation. Circular dichroism spectra confirm that these molecules adopt the A form under conditions of reduced water activity. UVRR results, obtained under similar conditions, suggest that the transition involves a series of intermediate forms between A and B. Cooperative and distinct transitions were observed for the bases and the sugars. Independent MD simulations on d(CGCGAATTCGCG)(2) show a spontaneous change from the A to B form in aqueous solution and describe a kinetic model that agrees well with UVRR results. Based on these observations, we predict that the mechanism of the transition involves a series of A/B hybrid forms and is sequential in nature, similar to previous crystallographic studies of derivatized duplexes. A simulation in which waters were restrained in the major groove of B DNA shows a rapid, spontaneous change from B to A at reduced water activity. These results indicate that a quasiergodic sampling of the solvent distribution may be a problem in going from B to A at reduced water activity in the course of an MD simulation.

Mechanism of nucleotide sensing in group II chaperonins.

Group II chaperonins mediate protein folding in an ATP‐dependent manner in eukaryotes and archaea. The binding of ATP and subsequent hydrolysis promotes the closure of the multi‐subunit rings where protein folding occurs. The mechanism by which local changes in the nucleotide‐binding site are communicated between individual subunits is unknown. The crystal structure of the archaeal chaperonin from Methanococcus maripaludis in several nucleotides bound states reveals the local conformational changes associated with ATP hydrolysis. Residue Lys‐161, which is extremely conserved among group II chaperonins, forms interactions with the γ‐phosphate of ATP but shows a different orientation in the presence of ADP. The loss of the ATP γ‐phosphate interaction with Lys‐161 in the ADP state promotes a significant rearrangement of a loop consisting of residues 160–169. We propose that Lys‐161 functions as an ATP sensor and that 160–169 constitutes a nucleotide‐sensing loop (NSL) that monitors the presence of the γ‐phosphate. Functional analysis using NSL mutants shows a significant decrease in ATPase activity, suggesting that the NSL is involved in timing of the protein folding cycle.

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Archaeal and eukaryotic cytosols contain group II chaperonins, which have a double-barrel structure and fold proteins inside a cavity in an ATP-dependent manner. The most complex of the chaperonins, the eukaryotic TCP-1 ring complex (TRiC), has eight different subunits, chaperone containing TCP-1 (CCT1–8), that are arranged so that there is one of each subunit per ring. Aspects of the structure and function of the bovine and yeast TRiC have been characterized, but studies of human TRiC have been limited. We have isolated and purified endogenous human TRiC from HeLa suspension cells. This purified human TRiC contained all eight CCT subunits organized into double-barrel rings, consistent with what has been found for bovine and yeast TRiC. The purified human TRiC is active as demonstrated by the luciferase refolding assay. As a more stringent test, the ability of human TRiC to suppress the aggregation of human γD-crystallin was examined. In addition to suppressing off-pathway aggregation, TRiC was able to assist the refolding of the crystallin molecules, an activity not found with the lens chaperone, α-crystallin. Additionally, we show that human TRiC from HeLa cell lysate is associated with the heat shock protein 70 and heat shock protein 90 chaperones. Purification of human endogenous TRiC from HeLa cells will enable further characterization of this key chaperonin, required for the reproduction of all human cells.

The group II chaperonin Mm-Cpn binds and refolds human γD crystallin

Chaperonins assist in the folding of nascent and misfolded proteins, though the mechanism of folding within the lumen of the chaperonin remains poorly understood. The archeal chaperonin from Methanococcus marapaludis, Mm‐Cpn, shares the eightfold double barrel structure with other group II chaperonins, including the eukaryotic TRiC/CCT, required for actin and tubulin folding. However, Mm‐Cpn is composed of a single species subunit, similar to group I chaperonin GroEL, rather than the eight subunit species needed for TRiC/CCT. Features of the β‐sheet fold have been identified as sites of recognition by group II chaperonins. The crystallins, the major components of the vertebrate eye lens, are β‐sheet proteins with two homologous Greek key domains. During refolding in vitro a partially folded intermediate is populated, and partitions between productive folding and off‐pathway aggregation. We report here that in the presence of physiological concentrations of ATP, Mm‐Cpn suppressed the aggregation of HγD‐Crys by binding the partially folded intermediate. The complex was sufficiently stable to permit recovery by size exclusion chromatography. In the presence of ATP, Mm‐Cpn promoted the refolding of the HγD‐Crys intermediates to the native state. The ability of Mm‐Cpn to bind and refold a human β‐sheet protein suggests that Mm‐Cpn may be useful as a simplified model for the substrate recognition mechanism of TRiC/CCT.

Nitric oxide reduces sickle hemoglobin polymerization: Potential role of nitric oxide-induced charge alteration in depolymerization

We previously demonstrated that inhaling nitric oxide (NO) increases the oxygen affinity of sickle red blood cells (RBCs) in patients with sickle cell disease (SCD). Our recent studies found that NO lowered the P(50) values of sickle hemoglobin (HbS) hemolysates but did not increase methemoglobin (metHb) levels, supporting the role of NO, but not metHb, in the oxygen affinity of HbS. Here we examine the mechanism by which NO increases HbS oxygen affinity. Because anti-sickling agents increase sickle RBC oxygen affinity, we first determined whether NO exhibits anti-sickling properties. The viscosity of HbS hemolysates, measured by falling ball assays, increased upon deoxygenation; NO treatment reduced the increment. Multiphoton microscopic analyses showed smaller HbS polymers in deoxygenated sickle RBCs and HbS hemolysates exposed to NO. These results suggest that NO inhibits HbS polymer formation and has anti-sickling properties. Furthermore, we found that HbS treated with NO exhibits an isoelectric point similar to that of HbA, suggesting that NO alters the electric charge of HbS. NO-HbS adducts had the same elution time as HbA upon high performance liquid chromatography analysis. This study demonstrates that NO may disrupt HbS polymers by abolishing the excess positive charge of HbS, resulting in increased oxygen affinity.

Real Time Monitoring of Sickle Cell Hemoglobin Fiber Formation by UV Resonance Raman Spectroscopy

In sickle cell hemoglobin, individual tetramers associate into long fibers as a consequence of the mutation at the beta6 position. In this study UV resonance Raman spectroscopy is used to monitor the formation of Hb S fibers in real time through aromatic amino acid vibrational modes. The intermolecular contact formed by the mutation site ((1)beta(1)6 Glu-->Val) of one tetramer and the (2)beta(2)85 Phe-(2)beta(2)88 Leu hydrophobic pocket on a different tetramer is observed by monitoring the increase in signal intensity of Phe vibrational modes as a function of time, yielding kinetic progress curves similar to those obtained by turbidity measurements. Comparison of individual spectra collected at early time points (<1000 s) show small Phe intensity changes, which are attributed to weak transient associations of Hb S tetramers during the initial stages of the polymerization process. At later times (1000-2000 s) Phe signal intensity steadily increases because of increasing hydrophobicity of local Phe environment, a consequence of forming more stable (1)beta(1)-(2)beta(2) contacts. Tyr and Trp vibrational modes monitor H-bond strength between critical residues at the alpha(1)beta(2) interface of individual tetramers. Kinetic progress curves generated from these signals exhibit two distinct transitions at 2040 and 7340 s. These transitions, which occur later in time than those detected either by turbidity (1560 s) or by Phe signal intensity (1680 s), are attributed to initial fiber formation and subsequent formation of larger assemblies, such as macrofibers or gels. These results provide molecular insight into the interactions governing Hb S fiber formation.

Group II archaeal chaperonin recognition of partially folded human γD-crystallin mutants.

The features in partially folded intermediates that allow the group II chaperonins to distinguish partially folded from native states remain unclear. The archaeal group II chaperonin from Methanococcus Mauripaludis (Mm‐Cpn) assists the in vitro refolding of the well‐characterized β‐sheet lens protein human γD‐crystallin (HγD‐Crys). The domain interface and buried cores of this Greek key conformation include side chains, which might be exposed in partially folded intermediates. We sought to assess whether particular features buried in the native state, but absent from the native protein surface, might serve as recognition signals. The features tested were (a) paired aromatic side chains, (b) side chains in the interface between the duplicated domains of HγD‐Crys, and (c) side chains in the buried core which result in congenital cataract when substituted. We tested the Mm‐Cpn suppression of aggregation of these HγD‐Crys mutants upon dilution out of denaturant. Mm‐Cpn was capable of suppressing the off‐pathway aggregation of the three classes of mutants indicating that the buried residues were not recognition signals. In fact, Mm‐Cpn recognized the HγD‐Crys mutants better than (wild‐type) WT and refolded most mutant HγD‐Crys to levels twice that of WT HγD‐Crys. This presumably represents the increased population or longer lifetimes of the partially folded intermediates of the mutant proteins. The results suggest that Mm‐Cpn does not recognize the features of HγD‐Crys tested—paired aromatics, exposed domain interface, or destabilized core—but rather recognizes other features of the partially folded β‐sheet conformation that are absent or inaccessible in the native state of HγD‐Crys.

How Do Group Ii Chaperonins Distinguish their Partially Folded Substrates from the Native States

The features in partially folded intermediates that allow the group II chaperonins to distinguish partially folded from native states remain unclear. The Archaeal group II chaperonin Mm-Cpn assists the in vitro refolding of the well-characterized β-sheet lens protein human γD-Crystallin (HγD-Crys). The buried cores of this Greek key conformation as well as the domain interface includes a variety of side chains which might be exposed in partially folded intermediates. We sought to assess whether particular features buried in the native state, and thus absent from the native protein surface, might be serving as recognition signals. The features tested were a) paired aromatic side chains; b) side chains in the interface between the duplicated domains of HγD-Crys, and c) side chains in the buried core which result in congenital cataract when substituted. We tested the Mm-Cpn suppression of aggregation of HγD-Crys mutants of these residues, during refolding after dilution out of denaturant. Mm-Cpn was capable of suppressing the off-pathway aggregation of the three classes of mutant proteins indicating that the buried residues were not recognition signals. In fact Mm-Cpn refolded most mutant HγD-Crys to levels twice that of WT HγD-Crys. This presumably represents the increased population or longer lifetimes of the partially folded intermediates of the destabilized mutant proteins. The results suggest that Mm-Cpn does not recognize paired aromatic residues, exposed domain interface, or destabilized core - but rather recognizes other features of the partially folded β-sheet, such as an exposed backbone, that are absent or inaccessible in the native state off HγD-Crys. Supported by NIH EY15834 and EY016525.