Equatorial Domain Research Articles

The Crystal Structure of the Outer Membrane Protein VceC from the Bacterial Pathogen Vibrio cholerae at 1.8 Å Resolution

Multidrug resistance in Gram-negative bacteria arises in part from the activities of tripartite drug efflux pumps. In the pathogen Vibrio cholerae, one such pump comprises the inner membrane proton antiporter VceB, the periplasmic adaptor VceA, and the outer membrane channel VceC. Here, we report the crystal structure of VceC at 1.8 A resolution. The trimeric VceC is organized in the crystal lattice within laminar arrays that resemble membranes. A well resolved detergent molecule within this array interacts with the transmembrane beta-barrel domain in a fashion that may mimic protein-lipopolysaccharide contacts. Our analyses of the external surfaces of VceC and other channel proteins suggest that different classes of efflux pumps have distinct architectures. We discuss the implications of these findings for mechanisms of drug and protein export.

The 13 Å Structure of a Chaperonin GroEL–Protein Substrate Complex by Cryo-electron Microscopy

The 13 angstroms resolution structures of GroEL bound to a single monomer of the protein substrate glutamine synthetase (GS(m)), as well as that of unliganded GroEL have been determined from a heterogeneous image population using cryo-electron microscopy (cryo-EM) coupled with single-particle image classification and reconstruction techniques. We combined structural data from cryo-EM maps and dynamic modeling, taking advantage of the known X-ray crystallographic structure and normal mode flexible fitting (NMFF) analysis, to describe the changes that occur in GroEL structure induced by GS(m) binding. The NMFF analysis reveals that the molecular movements induced by GS(m) binding propagate throughout the GroEL structure. The modeled molecular motions show that some domains undergo en bloc movements, while others show more complex independent internal movements. Interestingly, the substrate-bound apical domains of both the cis (GS(m)-bound ring) and trans (the opposite substrate-free ring) show counterclockwise rotations, in the same direction (though not as dramatic) as those documented for the ATP-GroEL-induced structure changes. The structural changes from the allosteric substrate protein-induced negative cooperativity between the GroEL rings involves upward concerted movements of both cis and trans equatorial domains toward the GS(m)-bound ring, while the inter-ring distances between the heptamer contact residues are maintained. Furthermore, the NMFF analysis identifies the secondary structural elements that are involved in the observed approximately 5 angstroms reduction in the diameter of the cavity opening in the unbound trans ring. Understanding the molecular basis of these substrate protein-induced structural changes across the heptamer rings provides insight into the origins of the allosteric negative cooperative effects that are transmitted over long distances (approximately 140 angstroms).

Impaired E-cadherin expression in human spermatozoa in a male factor infertility subset signifies E-cadherin-mediated adhesion mechanisms operative in sperm–oolemma interactions

Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell–cell adhesion in virtually all solid tissues of multicellular organisms. We have examined the presence of a cadherin on spermatozoon and its possible involvement in sperm–oocyte interaction. Spermatozoa from fertile human subjects showed the presence of E-cadherin on its head domains, detectable only after permeabilization of the surface membranes. On the contrary, spermatozoa from oligozoospermic subjects did not possess E-cadherin on their principal acrosomal and equatorial domains. Immunoprecipitation and Western blot studies also showed the presence of E-cadherin in spermatozoa from fertile males and its absence in oligozoospermic males. Using RT-PCR, we detected E-cadherin message in the round cells of fertile males, which was absent in the cells from oligozoospermic males. The presence of anti-E-cadherin antibody brought about quantitative reduction in the sperm–oocyte binding in vitro. These observations indicate the possibility of the interplay of a cadherin-dependent homophilic and/or heterophilic adhesion interaction between spermatozoa and oocyte during fertilization. The absence of a key adhesion molecule in a human male infertility disorder points towards genetic defects causing failure in gamete interactions.

A Nonconserved Carboxy-Terminal Segment of GroEL Contributes to Reaction Temperature

The role of the C-terminal segment of the GroEL equatorial domain was analyzed. To understand the molecular basis for the different active temperatures of GroEL from three bacteria, we constructed a series of chimeric GroELs combining the C-terminal segment of the equatorial domain from one species with the remainder of GroEL from another. In each case, the foreign C-terminal segment substantially altered the active temperature range of the chimera. Substitution of L524 of Escherichia coli GroEL with the corresponding residue (isoleucine) from psychrophilic GroEL resulted in a GroE with approximately wild-type activity at 25 degrees C, but also at 10 degrees C, a temperature at which wild-type E. coli GroE is inactive. In a detailed look at the temperature dependence of the GroELs, normal E. coli GroEL and the L524I mutant became highly active above 14 degrees C and 12 degrees C respectively. Similar temperature dependences were observed in a surface plasmon resonance assay of GroES binding. These results suggested that the C-terminal segment of the GroEL equatorial domain has an important role in the temperature dependence of GroEL. Moreover, E. coli acquired the ability to grow at low temperature through the introduction of cold-adapted chimeric or L524I mutant groEL genes.

Crystal Structures of the Group II Chaperonin from Thermococcus strain KS-1: Steric Hindrance by the Substituted Amino Acid, and Inter-subunit Rearrangement between Two Crystal Forms

The crystal structures of the group II chaperonins consisting of the α subunit with amino acid substitutions of G65C and/or I125T from the hyperthermophilic archaeum Thermococcus strain KS-1 were determined. These mutants have been shown to be active in ATP hydrolysis but inactive in protein folding. The structures were shown to be double-ring hexadecamers in an extremely closed form, which was consistent with the crystal structure of native α 8β 8-chaperonin from Thermoplasma acidophilum. Comparisons of the present structures with the atomic structures of the GroEL 14–GroES 7–(ADP) 7 complex revealed that the deficiency in protein-folding activity with the G65C amino acid substitution is caused by the steric hindrance of the local conformational change in an equatorial domain. We concluded that this mutant chaperonin with G65C substitution is deprived of the smooth conformational change in the refolding-reaction cycle. We obtained a new form of crystal with a distinct space group at a lower concentration of sulfate ion in the presence of nucleotide. The crystal structure obtained at the lower concentration of sulfate ion tilts outward, and has much looser inter-subunit contacts compared with those in the presence of a higher concentration of sulfate ion. Such subunit rotation has never been characterized in group II chaperonins. The crystal structure obtained at the lower concentration of sulfate ion tilts outward, and has much looser inter-subunit contacts compared with those in the presence of a higher concentration of sulfate ion.

Carbon dioxide along WOCE line A14: Water masses characterization and anthropogenic entry

The meridional WOCE line A14, just east of the South Atlantic Mid‐Atlantic Ridge, was surveyed during the austral summer of 1995 from 4°N to 45°S. Full‐depth profiles of pH, total alkalinity (TA), and total inorganic carbon (CT) were measured, allowing a test of the internal consistency of the CO2 system parameters. The correlation between CT measured and calculated from pH and TA was very good (r2 = 0.998), with an insignificant average difference of 0.1 ± 3.0 μmol kg−1 (n = 964 data). CO2 certified reference materials (CRMs) and a collection of selected samples subsequently analyzed at the Scripps Institution of Oceanography were used to assess the accuracy of our measurements at sea with satisfactory results. The three measured CO2 system variables were then used to identify the characteristic array of zonal flows throughout the South Atlantic intersected by A14. Equatorial, subequatorial, subtropical, and subantarctic domains were identified at the depth range of the surface water, South Atlantic Central Water (SACW), Antarctic Intermediate Water (AAIW), Upper Circumpolar Water (UCPW), North Atlantic Deep Water (NADW) and Antarctic Bottom Water (AABW). The nonconservative CO2 system parameters (pH, TA, CT) have been useful in identifying the transition from aged subequatorial to ventilated subtropical surface, central and intermediate waters. They have been identified as good tracers of the zonal circulation of NADW, with marked flows at the equator, 13°S, and 22°S (the “Namib Col Current”) and the sharp transition from UNADW to UCPW at 23°S. The anthropogenic CO2 inventory (CANT) was estimated and compared with CFC‐derived apparent ages for different water masses along A14. The anthropogenic entry reached maximum in the relatively young and ventilated subantarctic and subtropical domains where AAIW was the most efficient CO2 trap. The calculated annual rate of CANT entry by AAIW was 0.82 μmol kg−1 y−1, in agreement with the annual rate estimated from the equilibrium between the atmospheric pCO2 increase and the upper mixed layer.

Annealing function of GroEL: structural and bioinformatic analysis

The Escherichia coli chaperonin system, GroEL–GroES, facilitates folding of substrate proteins (SPs) that are otherwise destined to aggregate. The iterative annealing mechanism suggests that the allostery-driven GroEL transitions leading to changes in the microenvironment of the SP constitutes the annealing action of chaperonins. To describe the molecular basis for the changes in the nature of SP–GroEL interactions we use the crystal structures of GroEL (T state), GroEL–ATP (R state) and the GroEL–GroES–(ADP)7 (R″ state) complex to determine the residue-specific changes in the accessible surface area and the number of tertiary contacts as a result of the T→R→R″ transitions. We find large changes in the accessible area in many residues in the apical domain, but relatively smaller changes are associated with residues in the equatorial domain. In the course of the T→R transition the microenvironment of the SP changes which suggests that GroEL is an annealing machine even without GroES. This is reflected in the exposure of Glu386 which loses six contacts in the T→R transition. We also evaluate the conservation of residues that participate in the various chaperonin functions. Multiple sequence alignments and chemical sequence entropy calculations reveal that, to a large extent, only the chemical identities and not the residues themselves important for the nominal functions (peptide binding, nucleotide binding, GroES and substrate protein release) are strongly conserved. Using chemical sequence entropy, which is computed by classifying aminoacids into four types (hydrophobic, polar, positively charged and negatively charged) we make several new predictions that are relevant for peptide binding and annealing function of GroEL. We identify a number of conserved peptide binding sites in the apical domain which coincide with those found in the 1.7 Å crystal structure of ‘mini-chaperone’ complexed with the N-terminal tag. Correlated mutations in the HSP60 family, that might control allostery in GroEL, are also strongly conserved. Most importantly, we find that charged solvent-exposed residues in the T state (Lys 226, Glu 252 and Asp 253) are strongly conserved. This leads to the prediction that mutating these residues, that control the annealing function of the SP, can decrease the efficacy of the chaperonin function.

Seasonal Migration of Monsoons between the Northern and Southern Hemisphere as Revealed from Equatorially Symmetric and Asymmetric OLR Data.

The climatological pentad mean OLR data are partitioned into symmetric component OLR´, and asymmetric component OLR´´, with reference to the equator. Objective criteria are then introduced to define the intensity, center and areal extent of strong convections with OLR´ (OLR´´) of less than 220 (-20) Wm-2 over the equatorial (subtropical) domains, and seasonal migration of monsoons between the two hemispheres is investigated. Over the equatorial continents, such as Africa and South America, OLR´ exhibits a common feature of distinct semi-annual standing oscillations, while OLR´´ is of annual standing oscillation character that is peculiar to the monsoon system. Coexistent of two standing oscillations of different periodicity implies occurrence of a systematic cross-equatorial propagation of strong convections twice a year, northward during the spring transition, and southward in fall. The manner in which the monsoon transits from one hemisphere to another is distinctly different between the Indian Ocean and Western Pacific. This is associated with difference in the systematic equatorial basic flow which is, to a large extent, regulated by equatorial convections occurring over the maritime continent. Eastward (westward) of the maritime continent around 125°E, is a Kelvin (Rossby)-type basic flow regime; that is subject to significant annual variations. During late fall to early winter over the equatorial Western Pacific is a very pronounced Kelvin-type basic flow enhanced by northerly surges emanating out of the Siberian and North Pacific high pressure systems. The symmetric Kelvin flow acts as a bridge through which convections migrate southward from the WNPM (Western North Pacific Monsoon) to WSPM (Western South Pacific Monsoon) domain by crossing the equator in November and December. Gradual weakening of the Kelvin-type flow regime after winter inhibits northward return journey of convections from WSPM to WNPM in April and May. During spring and early summer over the eastern equatorial Indian Ocean is a prominent symmetric Rossby-type basic flow, which serves as a medium for convections to propagate northward, although not well organized, from SIOM (South Indian Ocean Monsoon) to SEAM (Southeast Asian Monsoon). The fall transition is of different character. The asymmetric convection center established in SEAM migrates southward, until reaching the equator by October. Instead of penetrating into the South Indian Ocean, the convective center suddenly changes its direction and propagates eastward along the equator, reaching near Borneo by the end of the calender year.

Molecular mechanisms of chaperonin GroEL-GroES function.

The dynamics of the GroEL-GroES complex is investigated with a coarse-grained model. This model is one in which single-residue points are connected to other such points, which are nearby, by identical springs, forming a network of interactions. The nature of the most important (slowest) normal modes reveals a wide variety of motions uniquely dependent upon the central cavity of the structure, including opposed torsional rotation of the two GroEL rings accompanied by the alternating compression and expansion of the GroES cap binding region, bending, shear, opposed radial breathing of the cis and trans rings, and stretching and contraction along the protein assembly's long axis. The intermediate domains of the subunits are bifunctional due to the presence of two hinges, which are alternatively activated or frozen by an ATP-dependent mechanism. ATP binding stabilizes a relatively open conformation (with respect to the central cavity) and hinders the motion of the hinge site connecting the intermediate and equatorial domains, while enhancing the flexibility of the second hinge that sets in motion the apical domains. The relative flexibilities of the hinges are reversed in the nucleotide-free form. Cooperative cross-correlations between subunits provide information about the mechanism of action of the protein. The mechanical motions driven by the different modes provide variable binding surfaces and variable sized cavities in the interior to enable accommodation of a broad range of protein substrates. These modes of motion could be used to manipulate the substrate's conformations.

A 11.5 Å single particle reconstruction of GroEL using EMAN

Single-particle analysis has become an increasingly important method for structural determination of large macromolecular assemblies. GroEL is an 800 kDa molecular chaperone, which, along with its co-chaperonin GroES, promotes protein folding both in vitro and in the bacterial cell. EMAN is a single-particle analysis software package, which was first publicly distributed in 2000. We present a three-dimensional reconstruction of native naked GroEL to ∼11.5 Å performed entirely with EMAN. We demonstrate that the single-particle reconstruction, X-ray scattering data and X-ray crystal structure all agree well at this resolution. These results validate the specific methods of image restoration, reconstruction and evaluation techniques implemented in EMAN. It also demonstrates that the single-particle reconstruction technique and X-ray crystallography will yield consistent structure factors, even at low resolution, when image restoration is performed correctly. A detailed comparison of the single-particle and X-ray structures exhibits some small variations in the equatorial domain of the molecule, likely due to the absence of crystal packing forces in the single-particle reconstruction.

Crystal Structure of Chaperonin-60 from Paracoccus denitrificans

The crystal structure of chaperonin-60 from Paracoccus denitrificans (P.cpn60) has been determined at 3.2Å resolution by the molecular replacement method. Two heptameric rings of identical subunits of P.cpn60 in adjacent asymmetric units are stacked in a back-to-back manner and form a cylinder, as found in GroEL, cpn60 from Escherichia coli. With respect to the unliganded GroEL structure, each subunit of P.cpn60 tilts 2° outwards and the apical domain twists 4° counter-clockwise in the top view in a hinge-like manner, rendering the central hole 5Å wider. Despite the subunit tilts, both rings in P.cpn60 contact at two sites of the equatorial domain in the same way as in GroEL. Interactions between residues 434 and 434, and 463 and 463 observed in GroEL were not found in P.cpn60, and the interaction between 452 and 461 was weaker in P.cpn60 than in GroEL. The unique hydrogen bond between 468 and 471 was observed at the right site in P.cpn60, which could account for why the subunits tilt outwards. The contact surface area was reduced at the left site, which is similar to the observed changes in the GroEL structures induced by ATP binding. In general, inter-ring interactions in P.cpn60 were weakened, which is consistent with findings that P.cpn60 is observed in single-ring forms as well as in double-ring forms.

Gene Duplication and the Evolution of Group II Chaperonins: Implications for Structure and Function

Chaperonins are multisubunit protein-folding assemblies. They are composed of two distinct structural classes, which also have a characteristic phylogenetic distribution. Group I chaperonins (called GroEL/cpn60/hsp60) are present in Bacteria and eukaryotic organelles while group II chaperonins are found in Archaea (called the thermosome or TF55) and the cytoplasm of eukaryotes (called CCT or TriC). Gene duplication has been an important force in the evolution of group II chaperonins: Archaea possess one, two, or three homologous chaperonin subunit-encoding genes, and eight distinct CCT gene families (paralogs) have been described in eukaryotes. Phylogenetic analyses indicate that while the duplications in archaeal chaperonin genes have occurred numerous times independently in a lineage-specific fashion, the eight different CCT subunits found in eukaryotes are the products of duplications that occurred early and very likely only once in the evolution of the eukaryotic nuclear genome. Analyses of CCT sequences from diverse eukaryotic species reveal that each of the CCT subunits possesses a suite of invariant subunit-specific amino acid residues (“signatures”). When mapped onto the crystal structure of the archaeal chaperonin from Thermoplasma acidophilum, these signatures are located in the apical, intermediate, and equatorial domains. Regions that were found to be variable in length and/or amino acid sequence were localized primarily to the exterior of the molecule and, significantly, to the extreme tip of the apical domain (the “helical protrusion”). In light of recent biochemical and electron microscopic data describing specific CCT–substrate interactions, our results have implications for the evolution of subunit-specific functions in CCT.

Allostery and protein substrate conformational change during GroEL/GroES-mediated protein folding

Chaperonins are large, double-ring complexes that assist the folding to native form of a wide variety of proteins in the cytosol of all cells and in mitochondria and chloroplasts of eukaryotes. Advances in structural biology are revealing additional states of the chaperonin machine that represent essential steps in the protein folding reaction. At the same time, biochemical and biophysical approaches are addressing fundamental questions regarding the physical states and folding trajectories of substrate polypeptides within the privileged environment of the folding-active chaperonin complex. The Escherichia coli chaperonin system, comprising GroEL and its co-chaperonin GroES, is extensively studied as the paradigm for chaperonin-mediated, ATP-dependent protein folding. The GroEL-GroES-ADP7 crystal structure also provided important insights into the GroEL ATPase mechanism. The opening-out motion of GroEL subunits on binding GroES involves a downward rotation of the intermediate domain. GroEL is a 58-kDa protein that assembles into a cylindrical 800-kDa oligomer formed of two heptameric rings stacked back-to-back. Three domains are recognized in each subunit, designated equatorial, intermediate, and apical. The assembly is mainly held together by the equatorial domain, which is formed of the N- and C-terminal portions of the chain.

Excluded Volume Effects on the Refolding and Assembly of an Oligomeric Protein: GroEL, A CASE STUDY

We have studied the effect of macromolecular crowding reagents, such as polysaccharides and bovine serum albumin, on the refolding of tetradecameric GroEL from urea-denatured protein monomers. The results show that productive refolding and assembly strongly depends on the presence of nucleotides (ATP or ADP) and background macromolecules. Nucleotides are required to generate an assembly-competent monomeric conformation, suggesting that proper folding of the equatorial domain of the protein subunits into a native-like structure is essential for productive assembly. Crowding modulates GroEL oligomerization in two different ways. First, it increases the tendency of refolded, monomeric GroEL to undergo self-association at equilibrium. Second, crowding can modify the relative rates of the two competing self-association reactions, namely, productive assembly into a native tetradecameric structure and unproductive aggregation. This kinetic effect is most likely exerted by modifications of the diffusion coefficient of the refolded monomers, which in turn determine the conformational properties of the interacting subunits. If they are allowed to become assembly-competent before self-association, productive oligomerization occurs; otherwise, unproductive aggregation takes place. Our data demonstrate that the spontaneous refolding and assembly of homo-oligomeric proteins, such as GroEL, can occur efficiently (70%) under crowding conditions similar to those expected in vivo.

Circulation at the western boundary of the South and Equatorial Atlantic: Exchanges with the ocean interior

Data from a hydrographic section carried out in January-March 1994 offshore from the eastern coast of South America from 50S to 10N, are used to quantify the full-depth exchanges of water between the western boundary currents and the ocean interior. In the upper and intermediate layers, the westward transport associated with the southern branch of the South Equatorial Current was 49 Sv at the time of the cruise. The transports of the central and northern branches in the upper 200 m were 17 Sv and 12 Sv, respectively. After subtraction of the parts that recirculate in the subtropical, subequatorial, and equatorial domains, the fraction of the South Equatorial Current that effectively contributes to the warm water export to the North Atlantic is estimated at 18 Sv. The poleward boundary of the current southern branch is at 31S through the whole thickness of the subtropical gyre, but the latitude of the northern boundary varies from 7 degrees 30'S at the surface to 27S at 1400 m depth. The estimated latitude of its bifurcation into the Brazil Current and North Brazil Undercurrent also varies downward from about 14S at the surface to 28S at a depth of 600 m.In the North Atlantic Deep Water, eastward flows exceeding 10 Sv are observed at 3 degrees -4 degrees of latitude in both hemispheres, at 10S, and at 34S-30S. Between 4S and 17S, a net westward flow with an estimated transport of 19 Sv reinforces the southward deep western boundary current. Cyclonic circulations of Antarctic Bottom Water along the western boundaries of the Argentine and Brazil basins have amplitudes of 15 Sv and 13 Sv, respectively, exceeding those of the interbasin exchanges. The net alongshore transport of this water mass between the hydrographic section and the continental slope reverses to a southward direction from 13S to 27S, probably in relation with an eastward shift of the equatorward near-bottom boundary current at these latitudes.

Selection of peptides targeting the human sperm surface using random peptide phage display identify ligands homologous to ZP3.

Analysis of the surface architecture of human spermatozoa is a necessary step in the development of new approaches to contraception and resolving the causes of human infertility. In this study we have utilized phage display technology to identify peptides that bind with high affinity to the surface of human spermatozoa. Fifteen- and twelve-mer random peptide phage display libraries were screened against paraformaldehyde-fixed spermatozoa and a number of sperm-binding peptides were identified. One peptide, M6, displayed a high level of affinity for the sperm surface and showed sequence homology with a dominant human ZP3 epitope (hZP 25-33). This peptide bound preferentially to the equatorial and post acrosomal domains of the sperm head and exhibited contraceptive activity by virtue of its capacity to impair the fusion of acrosome-reacted spermatozoa with the vitelline membrane of the oocyte. A similar form of contraceptive activity was also observed within an unrelated peptide, K6, derived from screening the 12-mer library. These results indicate that phage display technology is a powerful tool for developing reagents capable of targeting the human sperm surface, providing insights into the composition of this structure and the identity of targets susceptible to contraceptive attack and pathological disruption.

How Ocean Vertical Mixing and Accumulation of Warm Surface Water Influence the “Sharpness” of the Equatorial Thermocline

This work utilizes an equatorial Pacific domain ocean general circulation model that achieves a sharp equatorial thermocline. The model has been developed for simulation of the El Nino-Southern Oscillation phenomenon and employs an integer power (IP) vertical mixing scheme formulation that is based on the observations of Peters, Gregg, and Toole. Model experiments are used to investigate the influence of warm surface water accumulation upon vertical mixing scheme performance. Straightforward techniques, applicable to any model, are used to diagnose the warm surface water accumulation. It is shown that if warm surface water is allowed to accumulate it can prevent the IP vertical mixing scheme from maintaining a sharp thermocline. Experiments reveal that, in the present model, the accumulation of the warm surface water occurs as a consequence of the model's zonal walls and their sponge layers. A parameterized wall heat transport scheme is then used to prevent the accumulation of warm surface water and this allows the IP scheme to maintain a sharp thermocline, but not the Pacanowski and Philander vertical mixing scheme. Results from a literature survey suggest that the problem of warm surface water accumulation may not be uncommon in other tropical ocean models with zonal walls and sponge layers, and that this may be one of the causes of the long-standing diffuse thermocline problem in these models. It is concluded that to achieve a sharp equatorial thermocline it is necessary for an ocean model to maintain realistic meridional heat transports and so correctly regulate the amount of warm surface water in the tropical ocean, and it is demonstrated that problems with vertical mixing and warm surface water accumulation can look confusingly similar. The ability of warm surface water accumulation to prevent a vertical mixing scheme from achieving a sharp thermocline may have influenced previous assessments of vertical mixing schemes.

A dynamic model for the allosteric mechanism of GroEL

GroEL-assisted protein folding is regulated by a cycle of large coordinated domain movements in the 14-subunit double-ring assembly. The transition path between the closed (unliganded) and the open (liganded) states, calculated with a targeted molecular dynamics simulation, shows the highly complex subunit displacements required for the allosteric transition. The early downward motion of the small intermediate domain induced by nucleotide binding emerges as the trigger for the larger movements of the apical and equatorial domains. The combined twisting and upward displacement of the apical domain determined for a single subunit is accommodated easily in the heptamer ring only if its opening is concerted. This is a major source of cooperative ligand binding within a ring. It suggests also that GroEL has evolved so that the motion required for heptamer cooperativity is encoded in the individual subunits. A calculated model for a di-cis 14-subunit assembly is found to be destabilized by strong steric repulsion between the equatorial domains of the two rings, the source of negative cooperativity. The simulation results, which indicate that transient interactions along the transition path are essential for GroEL function, provide a detailed structural description of the motions that are involved in the GroEL allosteric cycle.

Conformational Changes In GROEL Induced by a Protein Substrate

Abstract The GroEL/GroES chaperonin system of E. coli facilitates nucleotide dependent folding of select proteins. The structure of GroEL has been described by three-dimensional electron microscopy and at higher resolution by X-ray crystallography. The GroEL oligomer is a cylindrical tetradecamer composed of two heptameric rings of 57 kDa protein subunits, stacked back to back. Each subunit is comprised of two large domains, equatorial and apical, connected by a smaller intermediate domain. The intermediate “hinge” domain links the apical and equatorial domain and provides flexibility for en bloc rearrangement associated with nucleotide and GroES binding. Equatorial domains are responsible for interactions between the two heptamers and contain the ATPase activity of GroEL. Each ring of GroEL has a central cavity that is the binding site for denatured protein substrate. GroES and denatured substrate binding sites are located on the apical domains facing the central cavity.

Identifying the determinants in the equatorial domain of Buchnera GroEL implicated in binding Potato leafroll virus.

Luteoviruses avoid degradation in the hemolymph of their aphid vector by interacting with a GroEL homolog from the aphid's primary endosymbiotic bacterium (Buchnera sp.). Mutational analysis of GroEL from the primary endosymbiont of Myzus persicae (MpB GroEL) revealed that the amino acids mediating binding of Potato leafroll virus (PLRV; Luteoviridae) are located within residues 9 to 19 and 427 to 457 of the N-terminal and C-terminal regions, respectively, of the discontinuous equatorial domain. Virus overlay assays with a series of overlapping synthetic decameric peptides and their derivatives demonstrated that R13, K15, L17, and R18 of the N-terminal region and R441 and R445 of the C-terminal region of the equatorial domain of GroEL are critical for PLRV binding. Replacement of R441 and R445 by alanine in full-length MpB GroEL and in MpB GroEL deletion mutants reduced but did not abolish PLRV binding. Alanine substitution of either R13 or K15 eliminated the PLRV-binding capacity of the other and those of L17 and R18. In the predicted tertiary structure of GroEL, the determinants mediating virus binding are juxtaposed in the equatorial plain.