Steven G. Burston

The stability and hydrophobicity of cytosolic and mitochondrial malate dehydrogenases and their relation to chaperonin-assisted folding

mMDH and cMDH are structurally homologous enzymes which show very different responses to chaperonins during folding. The hydrophilic and stable cMDH is bound by cpn60 but released by MG‐ATP alone, while the hydrophobic and unstable mMDH requires both Mg‐ATP and cpn 10. Citrate equalises the stability of the native state of the two proteins but has no effect on the co‐chaperonin requirement, implying that hydrophobicity, and not stability, is the determining factor. The yield and rate of folding of cMDH is unaffected while that of mMDH is markedly increased by the presence of cpn60, cpn10 and Mg‐ATP. In 200 mM orthophosphate, chaperonins do not enhance the rate of folding of mMDH, but in low phosphate concentrations chaperonin‐assisted folding is 3–4‐times faster.

An ACP structural switch: conformational differences between the apo and holo forms of the actinorhodin polyketide synthase acyl carrier protein.

The actinorhodin (act) synthase acyl carrier protein (ACP) from Streptomyces coelicolor plays a central role in polyketide biosynthesis. Polyketide intermediates are bound to the free sulfhydryl group of a phosphopantetheine arm that is covalently linked to a conserved serine residue in the holo form of the ACP. The solution NMR structures of both the apo and holo forms of the ACP are reported, which represents the first high resolution comparison of these two forms of an ACP. Ensembles of twenty apo and holo structures were calculated and yielded atomic root mean square deviations of well‐ordered backbone atoms to the average coordinates of 0.37 and 0.42 Å, respectively. Three restraints defining the protein to the phosphopantetheine interface were identified. Comparison of the apo and holo forms revealed previously undetected conformational changes. Helix III moved towards helix II (contraction of the ACP), and Leu43 on helix II subtly switched from being solvent exposed to forming intramolecular interactions with the newly added phosphopantetheine side chain. Tryptophan fluorescence and S. coelicolor fatty acid synthase (FAS) holo‐synthase (ACPS) assays indicated that apo‐ACP has a twofold higher affinity (Kd of 1.1 μM) than holo‐ACP (Kd of 2.1 μM) for ACPS. Site‐directed mutagenesis of Leu43 and Asp62 revealed that both mutations affect binding, but have differential affects on modification by ACPS. Leu43 mutations in particular strongly modulate binding affinity for ACPS. Comparison of apo‐ and holo‐ACP structures with known models of the Bacillus subtilis FAS ACP–holo‐acyl carrier protein synthase (ACPS) complex suggests that conformational modulation of helix II and III between apo‐ and holo‐ACP could play a role in dissociation of the ACP–ACPS complex.

Elucidation of Steps in the Capture of a Protein Substrate for Efficient Encapsulation by GroE

We have identified five structural rearrangements in GroEL induced by the ordered binding of ATP and GroES. The first discernable rearrangement (designated T → R1) is a rapid, cooperative transition that appears not to be functionally communicated to the apical domain. In the second (R1 → R2) step, a state is formed that binds GroES weakly in a rapid, diffusion-limited process. However, a second optical signal, carried by a protein substrate bound to GroEL, responds neither to formation of the R2 state nor to the binding of GroES. This result strongly implies that the substrate protein remains bound to the inner walls of the initially formed GroEL·GroES cavity, and is not yet displaced from its sites of interaction with GroEL. In the next rearrangement (R2·GroES → R3·GroES) the strength of interaction between GroEL and GroES is greatly enhanced, and there is a large and coincident loss of fluorescence-signal intensity in the labeled protein substrate, indicating that there is either a displacement from its binding sites on GroEL or at least a significant change of environment. These results are consistent with a mechanism in which the shift in orientation of GroEL apical domains between that seen in the apo-protein and stable GroEL·GroES complexes is highly ordered, and transient conformational intermediates permit the association of GroES before the displacement of bound polypeptide. This ensures efficient encapsulation of the polypeptide within the GroEL central cavity underneath GroES.

Structure and malonyl CoA-ACP transacylase binding of streptomyces coelicolor fatty acid synthase acyl carrier protein.

Malonylation of an acyl carrier protein (ACP) by malonyl Coenzyme A-ACP transacylase (MCAT) is fundamental to bacterial fatty acid biosynthesis. Here, we report the structure of the Steptomyces coelicolor (Sc) fatty acid synthase (FAS) ACP and studies of its binding to MCAT. The carrier protein adopts an alpha-helical bundle structure common to other known carrier proteins. The Sc FAS ACP shows close structural homology with other fatty acid ACPs and less similarity with Sc actinorhodin (act) polyketide synthase (PKS) ACP where the orientation of helix I differs. NMR experiments were used to map the binding of ACP to MCAT. This data suggests that Sc FAS ACP interacts with MCAT through the negatively charged helix II of ACP, consistent with proposed models for ACP recognition by other FAS enzymes. Differential roles for residues at the interface are demonstrated using site-directed mutagenesis and in vitro assays. MCAT has been suggested, moreover, to participate in bacterial polyketide synthesis in vivo. We demonstrate that the affinity of the polyketide synthase ACP for MCAT is lower than that of the FAS ACP. Mutagenesis of homologous helix II residues on the polyketide synthase ACP suggests that the PKS ACP may bind to MCAT in a different manner than the FAS counterpart.

Characterisation of a GroEL Single-Ring Mutant that Supports Growth of Escherichia coli and Has GroES-Dependent ATPase Activity

Binding and folding of substrate proteins by the molecular chaperone GroEL alternates between its two seven-membered rings in an ATP-regulated manner. The association of ATP and GroES to a polypeptide-bound ring of GroEL encapsulates the folding proteins in the central cavity of that ring (cis ring) and allows it to fold in a protected environment where the risk of aggregation is reduced. ATP hydrolysis in the cis ring changes the potentials within the system such that ATP binding to the opposite (trans) ring triggers the release of all ligands from the cis ring of GroEL through a complex network of allosteric communication between the rings. Inter-ring allosteric communication thus appears indispensable for the function of GroEL, and an engineered single-ring version (SR1) cannot substitute for GroEL in vivo. We describe here the isolation and characterisation of an active single-ring form of the GroEL protein (SR-A92T), which has an exceptionally low ATPase activity that is strongly stimulated by the addition of GroES. Dissection of the kinetic pathway of the ATP-induced structural changes in this active single ring can be explained by the fact that the mutation effectively blocks progression through the full allosteric pathway of the GroEL reaction cycle, thus trapping an early allosteric intermediate. Addition of GroES is able to overcome this block by binding this intermediate and pulling the allosteric pathway to completion via mass action, explaining how bacterial cells expressing this protein as their only chaperonin are viable.

Toward a detailed description of the pathways of allosteric communication in the GroEL chaperonin through atomistic simulation.

GroEL, along with its coprotein GroES, is essential for ensuring the correct folding of unfolded or newly synthesized proteins in bacteria. GroEL is a complex, allosteric molecule, composed of two heptameric rings stacked back to back, that undergoes large structural changes during its reaction cycle. These structural changes are driven by the cooperative binding and subsequent hydrolysis of ATP, by GroEL. Despite numerous previous studies, the precise mechanisms of allosteric communication and the associated structural changes remain elusive. In this paper, we describe a series of all-atom, unbiased, molecular dynamics simulations over relatively long (50-100 ns) time scales of a single, isolated GroEL subunit and also a heptameric GroEL ring, in the presence and absence of ATP. Combined with results from a distance restraint-biased simulation of the single ring, the atomistic details of the earliest stages of ATP-driven structural changes within this complex molecule are illuminated. Our results are in broad agreement with previous modeling studies of isolated subunits and with a coarse-grained, forcing simulation of the single ring. These are the first reported all-atom simulations of the GroEL single-ring complex and provide a unique insight into the role of charged residues K80, K277, R284, R285, and E388 at the subunit interface in transmission of the allosteric signal. These simulations also demonstrate the feasibility of performing all-atom simulations of very large systems on sufficiently long time scales on typical high performance computing facilities to show the origins of the earliest events in biologically relevant processes.

The Influence of Chaperonins on Protein Folding

Molecular chaperones are proteins that promote the correct folding, assembly, and transport of other protein mo1ecules.l The most widely studied of the molecular chaperones are the chaperonins; these form a subgroup found in prokaryotic cells, in mitochondria, and in pla~tids.*-~ They are oligomeric proteins of high molecular weight, which work in conjunction with a smaller, also oligomeric, coprotein. One such chaperonin is cpn60 from Escherichia coli, the product of the gro-EL gene locus. This has a subunit mass of approximately 60,000 daltons, and the whole protein exists as an assembly of 14 subunits in a “double-doughnut” structure, each ring comprising 7 subunits. The coprotein is produced by thegro-ES locus and is termed cpnlO by virtue of its 10,000-dalton subunit molecular weight. CpnlO exists as a “singledoughnut” structure of 7 subunits.6 In conjunction, these proteins have been shown to aid the folding and/or assembly of bacteriophages, multimeric and monomeric protein molecule^.^^* They function by binding to unfolded or partially folded protein molecules and, by transducing the energy of ATP hydrolysis, increasing the yield of the natively assembled form.9 The mechanistic detail of this process remains undefined. Here we report on preliminary kinetic experiments that explore the interactions of cpn60 with (a) the folding intermediates of Bacillus stearothemophilus lactate dehydrogenase (LDH), (b) with ATP and an unreactive analogue (AMP-PNP), and (c) with the coprotein cpnl0. From these results with LDH we suggest a mechanism that explains the ability of chaperonins to improve the efficiency of protein folding in general.