Changes In Subunit Structure Research Articles

Modeling Group II Chaperonin “Heat Shock” Protein Structure: pH and temperature dependency Piyasi Ghosh1,2 Vivek Govind Kumar1,3 Mahmoud Moradi3 Ruben Michael Ceballos1,2,4 University of Arkansas, Cell and Molecular Biology Program University of Arkansas, Department of Biological Sciences University of Arkansas, Department of Chemistry and Biochemistry Arkansas Center for Space and Planetary Sciences Program

Group II chaperonins or “heat shock proteins” (HSPs) are expressed in hyperthermophilic archaea of the family Sulfolobaceae. Three chaperonins designated as HSPα, HSPβ, and HSPγ have been reported and serve as subunits in the formation of nonameric double-ring (18-mer) complexes. HSP complexes in nature function to protect cellular proteins (e.g., enzymes) during thermal shock and, perhaps, other stressors such as pH flux. HSPα and HSPβ are stoichiometrically favored in HSP complex formation, whereas the HSPγ subunit is more rarely found as a constituent subunit. In vitro, HSPα homomers, HSPβ homomers, and HSPα/HSPβ heteromers will form in the presence of ATP and divalent cations (e.g., Mg++). HSP complexes feature a central pore that leads to an internal cavity. Pore openings are found at both apical and basal extremities of the 18-mer complex. It has been suggested that HSPs have “opened” and “closed” states. Whether the pore is “opened” or “closed” is a function of conformational changes in the individual HSP subunits that lead to subunit rearrangements in the complex. Since the thermotolerant nature and protective function of HSP complexes is likely based on the ability of the system to open and close, it is of interest to understand the changes in subunit ultra-structure that mediate switching between to these two conformational states. In this study, molecular dynamics simulations were conducted test the relative stability of HSPα versus HSPβ and to determine how HSPα and HSPβ tertiary structure may be impacted by temperature and pH. SWISS-MODEL and PyMOL were used to generate the 3-D HSP structures. CHARMM-GUI solution builder was used to solvate the modeled structures in a “water box” as an energy minimization step prior to running simulations, which were performed in Nanoscale Molecular Dynamics (NAMD). Output was later visualized using Visual Molecular Dynamics (VMD). We demonstrate that HSP a-helical structures change conformation as pH drops from neutral to acidic. Our data suggest that changes in subunit structure under thermal shock lead to changes in pore diameter; however, it is unclear if pH flux influences opening and closing of the HSP complex pore. These results demonstrate a plausible biophysical mechanism by which group II chaperonin complexes change conformation while serving client proteins.

Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin.

Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing.

X-ray and molecular-dynamics studies onMycobacterium lepraesingle-stranded DNA-binding protein and comparison with other eubacterial SSB structures

The crystal structures of two forms of Mycobacterium leprae single-stranded DNA-binding protein (SSB) have been determined at 2.05 and 2.8 Å resolution. Comparison of these structures with the structures of other eubacterial SSBs indicates considerable variation in their quaternary association, although the DNA-binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but could be related to variation in protein stability. Molecular-dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype Escherichia coli SSB and the individual subunits of both proteins. Together, the X-ray studies and molecular-dynamics simulations yield information on the relatively rigid and flexible regions of the molecule and on the effect of oligomerization on flexibility. The simulations provide insight into the changes in subunit structure on oligomerization. They also provide insight into the stability and time evolution of the hydrogen bonds/water bridges that connect the two pairs of monomers in the tetramer.

The effects of high salt concentrations on the regulation of the substrate specificity of human recombinant deoxycytidine kinase.

Deoxycytidine kinase (dCK) is a salvage pathway enzyme with broad substrate specificity that can phosphorylate both pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. The kinetic behaviour of dCK is complex with saturation curves showing negative cooperativity. In this study, we have expressed and affinity purified recombinant dCK, using the pET 9d vector system with a histidine tag-sequence and a thrombin cleavage site fused to the N-terminus of the dCK coding sequence. The His-tagged protein showed essentially the same kinetic properties as the protease cleaved protein and the purified protein isolated from human spleen. However, addition of 0.2-0.4 M NaCl during the dCK reaction caused a stimulation of 2'-deoxycytidine (dCyd), and the antileukemic analog 2-chlorodeoxyadenosine (CldAdo) phosphorylation, but an inhibition of the 2'-deoxyguanosine (dGuo) phosphorylation, both with His-tagged and protease cleaved dCK. The negative cooperativity observed with dCyd was eliminated by the presence of 0.4 M NaCl so that the Hill coefficient changed from 0.6 to 1.4. In contrast, dGuo phosphorylation that initially followed Michaelis-Menten kinetics showed negative cooperativity after addition of 0.4 NaCl. The alterations in kinetic properties were not accompanied by any apparent changes in subunit structure as revealed by gel filtration. The major form of dCK eluted in the position corresponding to a dimer in the presence or absence of salt, but a minor fraction of dCK, eluting in the position of a tetramer, was diminished in the presence of salt. The mechanism for the effects of 0.4 M NaCl on dCK kinetic behaviour is not known but it is most likely due to alterations in the conformation of the active site of the enzyme. The effects described here also may explain some of the discrepancies reported in the literature on the substrate specificity of this complex enzyme.

A Subunit Interaction in Chloroplast ATP Synthase Determined by Genetic Complementation between Chloroplast and Bacterial ATP Synthase Genes

F1F0-ATP synthases utilize protein conformational changes induced by a transmembrane proton gradient to synthesize ATP. The allosteric cooperativity of these multisubunit enzymes presumably requires numerous protein-protein interactions within the enzyme complex. To correlate known in vitro changes in subunit structure with in vivo allosteric interactions, we introduced the beta subunit of spinach chloroplast coupling factor 1 ATP into a bacterial F1 ATP synthase. A cloned atpB gene, encoding the complete chloroplast beta subunit, complemented a chromosomal deletion of the cognate uncD gene in Escherichia coli and was incorporated into a functional hybrid F1 ATP synthase. The cysteine residue at position 63 in chloroplast beta is known to be located at the interface between alpha and beta subunits and to be conformationally coupled, in vitro, to the nucleotide binding site > 40 A away. Enlarging the side chain of chloroplast coupling factor 1 beta residue 63 from Cys to Trp blocked ATP synthesis in vivo without significantly impairing ATPase activity or ADP binding in vitro. The in vivo coupling of nucleotide binding at catalytic sites to transmembrane proton movement may thus involve an interaction, via conformational changes, between the amino-terminal domains of the alpha and beta subunits.

Limited degradation of vitellin and egg-specific protein in Bombyx eggs during embryogenesis

The metabolic fate of yolk proteins was followed by examining changes in subunit structure and immunochemical properties during embryogenesis in Bombyx mori. SDS-polyacrylamide gel electrophoresis of extracts from newly laid eggs showed eight major polypeptides that corresponded to the subunits of three yolk proteins, vitellin (Vtn), egg-specific protein (ESP) and the 30 kDa proteins. During embryogenesis, the peptides corresponding to Vtn and ESP decreased, and four new peptides (96, 55, 46 and 36 kDa) appeared at specific stages, but they disappeared suddenly before larval hatching. Vtn and ESP peptides stained with dyes specific for lipids (Sudan black), for sugars (Schiff's PAS reagent) and for phosphate (methyl green), but the newly appearing peptides stained only with Schiff's PAS reagent. Using a specific antiserum bound-protein A affinity column, two of the new peptides (55 and 36 kDa) were shown to be derived from ESP, and the other two peptides (96 and 46 kDa) from Vtn, suggesting that limited proteolysis of ESP and Vtn occurs during embryogenesis. In contrast, the 30 kDa proteins remained almost unchanged throughout the egg stage.

Ligand-induced asymmetry as observed through fluorophore rotations and free energy couplings: application to neurophysin.

Changes that occur in subunit neurophysin structure upon ligand binding were explored by two methods. First, the thermal coefficient of the viscosity around the subunit tyrosine was monitored, which yields information on the environmental flexibility and free rotational space of the fluorophore. Initially, it was determined that the environmental flexibility and the free space around each subunit tyrosine are unperturbed upon dimerization. Binding of the tripeptide analogue of oxytocin causes the once homologous environments of the subunit tyrosines to become drastically different such that one moves onto a closely packed environment whereas the other moves into a region of larger free space. Even though the subunits as seen by each tyrosine are very different, the specific binding sites as seen by the ligands are similar. It was also found that ligand binding is stabilized by ring stacking and that energy transfer occurs between the tyrosine of the ligand and the neurophysin subunit tyrosine. Second, changes in subunit structure upon ligation were also followed by the determination of the order of free energy coupling between ligand binding and oligomerization, which tells how each ligand affects the subunit affinity. Since the binding of ligand is cooperative and induces dimer formation, there is second-order coupling between ligand binding and dimerization and the binding of the second ligand is responsible for the increase in subunit affinity.