Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry 39, 13336-13343]. Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-3H]-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141. Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.     

In vitro hybridization and separation of hybrids of human adenylosuccinate lyase from wild-type and disease-associated mutant enzymes

Human adenylosuccinate lyase (ASL) deficiency is an inherited metabolic disease in which the majority of the patients are compound heterozygotes for the mutations that occur in the ASL gene. Starting with purified wild-type (WT) and single-mutant human ASL, we generated in vitro hybrids that mimic compound heterozygote ASL. For this study, we used His-tagged WT/non-His-tagged WT, His-tagged WT/non-His-tagged R396C, His-tagged WT/non-His-tagged R396H, His-tagged R194C/non-His-tagged R396C, and His-tagged L311V/non-His-tagged R396H enzyme pairs. We generated various hybrids by denaturing pairs of enzymes in 1 M guanidinium chloride and renaturing them by removing the denaturant. The hybrids were separated on a nickel-nitrilotriacetic acid-agarose column based on the number of His tags present in the enzyme tetramer. Analytical ultracentrifuge data indicate that the hybrids have predominant amounts of heterotetramers. Analysis of the V(max) values of the hybrids indicates that most of the subunits behave independently; however, the hybrid tetramers retain weak positive cooperativity, indicating that there is some interaction between the different subunit types. The interactions between WT and mutant subunits may be advantageous to the parents of ASL deficient patients, while the interactions between some mutant subunits may assist heterozygote ASL deficient patients.     

Effect of a new non-cleavable substrate analog on wild-type and serine mutants in the signature sequence of adenylosuccinate lyase of Bacillus subtilis and Homo sapiens

Adenylosuccinate lyase (ASL) catalyzes two beta-elimination reactions in purine biosynthesis, leading to the question of whether the two substrates occupy the same or different active sites. Kinetic studies of Bacillus subtilis and human ASL with a new substrate analog, adenosine phosphonobutyric acid, 2'(3'), 5'-diphosphate (APBADP), show that it acts as a competitive inhibitor with respect to either substrate (K(I) approximately 0.1 microM), indicating that the two substrates occupy the same active site. Binding studies show that both the B. subtilis and human ASLs bind up to 4 mol of APBADP per mole of enzyme tetramer and that both enzymes exhibit cooperativity: negative for B. subtilis ASL and positive for human ASL. Mutant B. subtilis ASLs, with replacements for residues previously identified as critical for catalysis, bind the substrate analog similarly to wild-type ASL. Two serines in a flexible loop of ASL have been proposed to play roles in catalysis because they are close to the substrate in the crystal structure of Escherichia coli ASL. We have now mutated the corresponding serines to alanines in B. subtilis and human ASL to evaluate their involvement in enzyme function. Kinetic data reveal that human Ser(289) and B. subtilis Ser(262) and Ser(263) are essential for catalysis, while the ability of these Ser mutants to bind APBADP suggests that they do not contribute to substrate affinity. Although these serines are not visible in the crystal structure of human adenylosuccinate lyase complexed with substrate or products (PDB #2VD6), they may be interacting with the active sites.     

α-Crystallin protects human arginosuccinate lyase activity under freeze-thaw conditions

Argininosuccinate lyase (ASL) catalyzes the conversion of argininosuccinate into arginine and fumarate, a key step in the biosynthesis of urea and arginine. ASL is a tetrameric enzyme but it dissociates into inactive dimers under low temperature conditions. This study investigates the inactivation process under low temperature conditions. Inactivation was caused by dissociation of tetrameric ASL into dimers, with increased exposure of hydrophobic areas without disturbance of the secondary structure or the microenvironment surrounding the key tryptophan residues. Most activity was retained when temperatures were changed at a rate of >1 °C/min, whilst freezing or thawing more slowly resulted in greater loss of activity. Inactivation was reduced by inclusion of α-crystallin, a structural protein found in ocular lenses and a member of the small heat-shock protein family, by stabilization of the ASL quaternary structure. In addition, α-crystallin was able to restore the function of ASL that had been inactivated by slow freezing and thawing. The effect of α-crystallin was similar to that of bovine serum albumin, suggesting that both proteins exerted their effects by hydrophobic interactions. α-Crystallin therefore acts as a cryo-preservative that protects ASL activity during freezing and thawing.     

Disruption of a salt bridge dramatically accelerates subunit exchange in duck delta2 crystallin

Intragenic complementation is a unique property of oligomeric enzymes with which to study subunit-subunit interactions. Complementation occurs when different subunits, each possessing distinct mutations that render the individual homomutant proteins inactive, interact to form a heteromutant protein with partial recovery of activity. In this paper, complementation events between human argininosuccinate lyase (ASL) and its homolog, duck delta2 crystallin, were characterized. Different active site mutants in delta2 crystallin complement by the regeneration of native-like active sites as reported previously for ASL. The complementarity of the ASL and delta2 crystallin subunit interfaces was illustrated by the in vivo formation of active hybrid tetramers from inactive ASL and inactive delta2 crystallin mutants. Subunits of both ASL and delta2 crystallin do not dissociate and reassociate in vitro at room temperature, even after 6 days of incubation, indicating that the multimerization interface is very strong. However, disruption of a salt bridge network in the tetrameric interface of delta2 crystallin caused a drastic acceleration of subunit dissociation. Double mutants combining these interface mutants with active site mutants of delta2 crystallin were able to dissociate and reassociate to form active tetramers in vitro within hours. These results suggest that exchange of subunits may occur without unfolding of the monomer. Intragenic complementation in these interface mutants occurs by reintroducing the native salt bridge interaction upon hetero-oligomerization. Our studies demonstrate the value of intragenic complementation as a tool for investigating subunit-subunit interactions in oligomeric proteins.     

Characterization of argininosuccinate lyase (EC 4.3.2.1) from Chlamydomonas reinhardtii.

We have isolated and characterized argininosuccinate lyase (ASL; EC 4.3.2.1) from the photosynthetic green alga, Chlamydomonas reinhardtii. The general properties of Chlamydomonas ASL are very similar to those described previously for ASLs from phylogenetically diverse organisms. The algal ASL has a native Mr, determined by gel-filtration chromatography, of 218,000 +/- 25,000, and a pI of 5.4-5.6. The Km for argininosuccinate at 37 degrees C and pH 7.5 is 0.26 mM. The subunit Mr of Chlamydomonas ASL is approx. 50,000, determined by SDS/polyacrylamide-gel electrophoresis, in contrast with a previously reported value of 39,000. Rabbit antisera prepared against the Mr-50,000 protein completely abolished ASL activity in vitro. In contrast, serum prepared against the Mr-39,000 protein was ineffective in inhibiting ASL activity. Despite the general similarity of the physical properties of Chlamydomonas ASL and those of other ASLs, antiserum raised against the algal ASL did not cross-react with ASL preparations from Escherichia coli, Saccharomyces cerevisiae or bovine liver.     

Modifying the oligomeric state of cyclic amidase and its effect on enzymatic catalysis

A group of cyclic amidases, including hydantoinase, allantoinase, dihydropyrimidinase, and dihydroorotase, catalyze the reversible hydrolysis of cyclic ureides, such as 5-monosubstituted hydantoins and dihydropyrimidines. These four enzymes carry hydrophobic patches to form dimers. With the exception of dihydroorotase, these enzymes are further dimerized to form tetramers by hydrophobic interactions. This leads us to speculate that the hydrophobic interaction domain may be a significant factor in the catalytic property of these oligomeric cyclic amidases, for which activities are not allosterically regulated. We generated a dimeric D-hydantoinase by mutating five residues in the hydrophobic alpha-helical interface of a tetramer and analyzed the kinetic properties of the dimeric form of D-hydantoinase. The specific activity of the dimeric D-hydantoinase corresponds to 5.3% of the activity of tetrameric D-hydantoinase. This low specific activity of the dimeric D-hydantoinase indicates that the dimeric interaction to form a tetramer has a significant effect on the catalytic activity of this non-allosteric tetramer.     

Human platelet factor 4 subunit association/dissociation thermodynamics and kinetics

Platelet factor 4 (PF4) monomers (7800 daltons) form dimers and tetramers in varying molar ratios under certain solution conditions [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469]. The presence of a simplified aromatic region (one Tyr and two His) and resolved monomer, dimer, and tetramer Y60 3,5 ring proton resonances makes study of PF4 aggregate association/dissociation thermodynamics and kinetics possible. PF4 protein subunit association/dissociation equilibrium thermodynamic parameters have been derived by 1H NMR (500MHz) resonance line-fitting analysis of steady-state Y60 3,5 ring proton resonance monomer-dimer-tetramer populations as a function of temperature from 10 to 40 degrees C. Below 10 degrees C and above 40 degrees C, resonance broadening and overlap severely impaired analysis. Enthalpic and entropic contributions to dimer association Gibb's free energy [-5.1 kcal/mol (30 degrees C)] are +2.5 +/- 1 kcal/mol and +26 +/- 7 eu, respectively, and for tetramer association Gibb's free energy [-5.7 kcal/mol (30 degrees C)], they are -7.5 +/- 1 kcal/mol and -7 +/- 3 eu, respectively. These thermodynamic parameters are consistent with low dielectric medium electrostatic/hydrophobic interactions governing dimer formation and hydrogen bonding governing tetramer formation. Association/dissociation kinetic parameters, i.e., steady-state jump rates, have been derived from exchange-induced line-width increases and from 1H NMR (500 MHz) saturation-transfer and spin-lattice (Tl) relaxation experiments. From dissociation jump rates and equilibrium constants, association rate constants were estimated. For dimer and tetramer equilibria at 30 degrees C, unimolecular dissociation rate constants are 35 +/- 10 s-1 for dimer dissociation and 6 +/- 2 s-1 for tetramer dissociation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion of the noncooperative Bacillus subtilis aspartate transcarbamoylase into a cooperative enzyme by a single amino acid substitution.

Allosteric enzymes are part of a unique class of enzymes which regulate metabolic pathways. On the molecular level, allosteric regulation is the result of interactions between discrete binding sites on the enzyme. In order to accommodate these multiple binding sites, allosteric enzymes have evolved with oligomeric quaternary structures. However, only a few oligomeric enzymes are known to have regulatory interactions between binding sites. Is regulatory activity an inherent property of oligomeric enzymes? The trimeric Bacillus subtilis aspartate transcarbamoylase catalyzes the first committed step of the pyrimidine biosynthetic pathway and is not known to be a regulatory enzyme. When an alanine residue is substituted for the active-site residue Arg-99 by site-specific mutagenesis, the regulatory activity of homotropic substrate cooperativity (Hill coefficient of 1.5) is observed in the resulting mutant enzyme. These results suggest that homotropic regulation may have evolved by a relatively small number of mutations to an oligomeric enzyme.     

Characterization of intermediate species during the molecular assembly of aspartase

Molecular assembly of aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli was studied during the reversible denaturation. Although previous studies [Tokushige, M., Eguchi, G., and Hirata, F. (1977) Biochem. Biophys. Acta 480, 479-488] were unable to identify intermediate species during the course of reversible denaturation of aspartase, temperature-controlled HPLC and cross-linking with dimethyl suberimidate of the renaturation products showed that monomeric, dimeric and trimeric species occupied over 80% of the total oligomeric molecules below 13 degrees C; unlike the tetramer, these intermediates were without the activity. The degree of active tetramer formation was a linear function of the restoration of the activity below 18 degrees C, while above 23 degrees C, the activity regain was less than 70% restoration of tetrameric molecules. Upon examination by fluorescence spectroscopy, structural changes during reconstitution exhibited such complex kinetics that the rapid formation of structured oligomers proceeds first with a half-time of less than 10 sec, followed by slow subunit association. These results strongly suggest that the tetramer formation is an essential prerequisite, though not sufficient for the active enzyme.     

Modulation of human cytidine deaminase by specific aminoacids involved in the intersubunit interactions

An investigation was made of the role exerted by some residues supposed to be involved in the intersubunit interaction and also in the catalytic site of homotetrameric human cytidine deaminase (T-CDA). Attention was focused on Y33, Y60, R68, and F137 residues that are a part of a conserved region in most T-CDAs. Hence, a series of site-directed mutagenesis experiments was set up obtaining seven mutants: Y60G, Y33G, Y33F Y33S, F137A, R68G, and R68Q. Each active purified mutant protein was characterized kinetically, with a series of substrates and inhibitors, and the effect of temperature on enzyme activity and stability was also investigated. Circular dichroism (CD) experiments at different temperatures and in presence of small amounts of sodium dodecyl sulphate (SDS) were performed in all the soluble mutant CDAs. The results obtained by site-directed mutagenesis studies were compared to the crystallographic data of B. subtilis CDA and E. coli CDA and to molecular modeling studies previously performed on human CDA. The mutation of Y60 to glycine produced an enzyme with a more compact quaternary structure with respect to the wild-type; this mutation did not have a dramatic effect on cytidine deamination, but it slightly affected the binding with the substrate. None of the mutant CDAs in Y33 showed enzymatic activity; they existed only as monomers, indicating that this residue, located at the intersubunit interface, may be responsible for the correct folding of human CDA. The insertion of an alanine instead of phenylalanine at position 137 led to a soluble but completely inactive enzyme unable to form a tetramer, suggesting that F137 residue may be important for the assembling of the tetramer and also for the arrangement of the CDA active site. Finally, R68G and R68Q mutations revealed that the presence of the amino group seems to be important for the catalytic process but not for substrate binding, as already shown in B. subtilis CDA. The quaternary structure of R68Q was not affected by the mutation, as shown by the SDS-induced dissociation experiments and CD studies, whereas R68G dissociated very easily in presence of small amounts of SDS. These experiments indicated that in the human CDA, the side chain of arginine 68 involved in the catalytic process in one subunit active site might come from another subunit. The data obtained from these studies confirmed the presence of a complicated set of intersubunit interactions in the active site of human CDA, as shown in other T-CDAs.     

Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface

We have investigated factors affecting stability at the subunit-subunit interface of the dimeric enzyme 3-isopropylmalate dehydrogenase (IPMDH) from Bacillus subtilis. Site-directed mutagenesis was used to replace methionine 256, a key residue in the subunit interaction, with other amino acids. Thermal stability against irreversible inactivation of the mutated enzymes was examined by analyzing the residual activity after heat treatment. The mutations M256V and M256A increased thermostability by 2.0 and 6.0 degrees C, respectively, whereas the mutations M256L and M256I had no effect. Thermostability of the M256F mutated enzyme was 4.0 degrees C lower than that of the wild-type enzyme. To our surprise, increasing the hydrophobicity of residue 256 within the hydrophobic core of the enzyme resulted in a lower thermal stability. The mutated enzymes showed an inverse correlation between thermostability and the volume of the side chain at position 256. Based on the X-ray crystallographic structure of Escherichia coli IPMDH, the environment around M256 in the B.subtilis homolog is predicted to be sterically crowded. These results suggest that Met256 prevents favorable packing. Introduction of a smaller amino acid at position 256 improves the packing and stabilizes the dimeric structure of IPMDH. The van der Waals volume of the amino acid residue at the hydrophobic subunit interface is an important factor for maintaining the stability of the subunit-subunit interface and is not always optimized in the mesophilic IPMDH enzyme.     

Tiny TIM: a small, tetrameric, hyperthermostable triosephosphate isomerase

Comparative structural studies on proteins derived from organisms with growth optima ranging from 15 to 100 degrees C are beginning to shed light on the mechanisms of protein thermoadaptation. One means of sustaining hyperthermostability is for proteins to exist in higher oligomeric forms than their mesophilic homologues. Triosephosphate isomerase (TIM) is one of the most studied enzymes, whose fold represents one of nature's most common protein architectures. Most TIMs are dimers of approximately 250 amino acid residues per monomer. Here, we report the 2.7 A resolution crystal structure of the extremely thermostable TIM from Pyrococcus woesei, a hyperthermophilic archaeon growing optimally at 100 degrees C, representing the first archaeal TIM structure. P. woesei TIM exists as a tetramer comprising monomers of only 228 amino acid residues. Structural comparisons with other less thermostable TIMs show that although the central beta-barrel is largely conserved, severe pruning of several helices and truncation of some loops give rise to a much more compact monomer in the small hyperthermophilic TIM. The classical TIM dimer formation is conserved in P. woesei TIM. The extreme thermostability of PwTIM appears to be achieved by the creation of a compact tetramer where two classical TIM dimers interact via an extensive hydrophobic interface. The tetramer is formed through largely hydrophobic interactions between some of the pruned helical regions. The equivalent helical regions in less thermostable dimeric TIMs represent regions of high average temperature factor. The PwTIM seems to have removed these regions of potential instability in the formation of the tetramer. This study of PwTIM provides further support for the role of higher oligomerisation states in extreme thermal stabilisation.     

Two novel mutant human adenylosuccinate lyases (ASLs) associated with autism and characterization of the equivalent mutant Bacillus subtilis ASL

An Australian patient with autism was found to be heterozygous for two mutations in the gene encoding adenylosuccinate lyase (ASL), resulting in the protein mutations E80D and D87E. The patient's mother carried only the E80D mutation. The equivalent positions are 62 and 69 in Bacillus subtilis ASL. Although both human and B. subtilis enzymes normally have Asp at position 87 (or 69), the B. subtilis ASL has Ile and Asp at 62 and 65, respectively, whereas human ASL has Glu and Arg at the equivalent positions. We have constructed, expressed, and purified the double mutant I62E/D65R as a "humanized" normal B. subtilis enzyme to compare with enzymes with a single mutation at position 62 (I62D/D65R), at position 69 (I62E/D65R/D69E), or at both positions (I62D/D65R/D69E). V(max) for conversion of adenylosuccinate to AMP and fumarate is 0.57 micromol/min/mg for I62E/D65R, 0.064 micromol/min/mg for I62D/D65R, 0.27 micromol/min/mg for I62E/D65R/D69E, and 0.069 micromol/min/mg for I62D/D65R/D69E. The K(m) for adenylosuccinate is elevated in the X62D mutants, and I62D/D65R is the least stable of these ASLs at 37 degrees C. The CD spectra of mutant and wild type enzymes are similar; thus, there are no appreciable structural changes. Clearly the Asp(62) causes the most drastic effect on ASL function, whereas the Glu(69) mutation produces only modest change. These results emphasize the importance of expanding tests for ASL deficiency to individuals with developmental delay of any severity, including individuals with autistic spectrum disorder. This study further demonstrates the usefulness of the B. subtilis ASL as a model to mimic the defective enzyme in ASL deficiency.     

Numerical simulation of aldolase tetramer stability

A theoretical study of aldolase tetramer stability, conducted by finite difference Poisson-Boltzmann (FDPB) and modified Tanford-Kirkwood (MTK) techniques using the atomic coordinates of human aldolase, is described. A method for calculating the interaction energy between subunits is proposed. An analysis of the contribution of different energy terms to the stability and oligomeric equilibria (monomer ⇔ dimer ⇔ tetramer) of aldolase is made. It is shown that the loss of solvation energy and electrostatic interactions at very high and low pH-s destabilise the oligomers. These energy terms are compensated over a wide pH range by the stabilization energy due to hydrophobic interactions. It is shown that the aldolase tetramer is energetically more preferable than other oligomers in the pH range from 5 to 11. Subunit-subunit interactions within the tetramer suggest one dimeric form to be the most stable of the possible sub-parts. For this reason the tetramer can be thought of as a “dimer of dimers”. A comparison between our theoretical results and available experimental data shows that the dissociation of the aldolase tetramer below pH 3–4 cooperatively leads to acid denaturation. A second dissociation is predicted to occur at high pH (>12) in addition to the well known acidic dissociation. The analysis suggests that a mutation of His20 or Arg257 to a neutral residue could decrease the pH of the acidic dissociation by approximately 1 pH unit. Received: 16 February 1998 / Revised version: 8 April 1998 / Accepted: 19 April 1998     

Secondary substrate binding strongly affects activity and binding affinity of Bacillus subtilis and Aspergillus niger GH11 xylanases

The secondary substrate binding site (SBS) of Bacillus subtilis and Aspergillus niger glycoside hydrolase family 11 xylanases was studied by site-directed mutagenesis and evaluation of activity and binding properties of mutant enzymes on different substrates. Modification of the SBS resulted in an up to three-fold decrease in the relative activity of the enzymes on polymeric versus oligomeric substrates and highlighted the importance of several amino acids in the SBS forming hydrogen bonds or hydrophobic stacking interactions with substrates. Weakening of the SBS increased K(d) values by up to 70-fold in binding affinity tests using natural substrates. The impact that modifications in the SBS have both on activity and on binding affinity towards polymeric substrates clearly shows that such structural elements can increase the efficiency of these single domain enzymes on their natural substrates.     

Macromolecular binding equilibria in the lac repressor system: studies using high-pressure fluorescence spectroscopy

High hydrostatic pressure coupled with fluorescence polarization has been used to investigate protein subunit interactions and protein-operator association in lac repressor labeled with a long-lived fluorescent probe. On the basis of observation of a concentration-dependent sigmoidal decrease in the dansyl fluorescence polarization, we conclude that application of high hydrostatic pressure results in dissociation of the lac repressor tetramer. The 2-fold decrease in the rotational relaxation time and the high-pressure plateau are consistent with a tetramer to dimer transition. The volume change for tetramer dissociation to dimer is -82 +/- 5 mL/mol. The dissociation constant calculated from the data taken at 4.5 degrees C is 4.3 +/- 1.3 nM. The tetramer dissociation constant increases by a factor of 3 when the temperature is raised from 4.5 to 21 degrees C. A very small effect of inducer binding on the subunit dissociation is observed at 4.5 degrees C; the Kd increases from 4.5 to 7.1 nM. At 21 degrees C, however, inducer binding stabilizes the tetramer by approximately 0.8 kcal/mol. Pressure-induced monomer formation is indicated by the curves obtained upon raising the pH to 9.2. The addition of IPTG shifts the pressure transition to only slightly higher pressures at this pH, indicating that the stabilization of the tetramer by inducer is not as marked as that observed at pH 7.1. From the decrease in the polarization of the dansyl repressor-operator complexes, we also conclude that the application of pressure results their dissociation and that the volume change is large in absolute value (approximately 200 mL/mol). The lac repressor-operator complex is more readily dissociated upon the application of pressure than the tetramer alone, indicating that operator binding destabilizes the lac repressor tetramer.     

Co-reconstitution of phospholamban mutants with the Ca-ATPase reveals dependence of inhibitory function on phospholamban structure

Phospholamban (PLB), a 52-amino acid integral membrane protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum through PLB phosphorylation mediated by beta-adrenergic stimulation. Based on site-directed mutagenesis and coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in HEK 293 cells, and on spin label detection of PLB oligomeric state in lipid bilayers, it has been proposed that the monomeric form of PLB is the inhibitory species, and depolymerization of PLB is essential for its regulatory function. Here we have studied the relationship between PLB oligomeric state and function by in vitro co-reconstitution of PLB and its mutants with purified Ca-ATPase. We compared wild type-PLB (wt-PLB), which is primarily a pentamer on SDS-polyacrylamide gel electrophoresis (PAGE) at 25 degrees C, with two of its mutants, C41L-PLB and L37A-PLB, that are primarily tetramer and monomer, respectively. We found that the monomeric mutant L37A-PLB is a more potent inhibitor than wt-PLB, supporting the previous proposal that PLB monomer is the inhibitory species. On the other hand, C41L-PLB, which has a monomeric fraction comparable to that of wt-PLB on SDS-PAGE at 25 degrees C, has no inhibitory activity when assayed at 25 degrees C. However, at 37 degrees C, a 3-fold increase in the monomeric fraction of C41L-PLB on SDS-PAGE resulted in inhibitory activity comparable to that of wt-PLB. Upon increasing the temperature from 25 to 37 degrees C, no change in fraction monomer or inhibitory activity for wt-PLB and L37A-PLB was observed. Based on these results, the extent of inhibition of Ca-ATPase by PLB or its mutants appears to depend not only on the propensity of PLB to dissociate into monomers but also on the relative potency of the particular PLB monomer when interacting with the Ca-ATPase.     

SAS‐6 coiled‐coil structure and interaction with SAS‐5 suggest a regulatory mechanism in C. elegans centriole assembly

The centriole is a conserved microtubule‐based organelle essential for both centrosome formation and cilium biogenesis. Five conserved proteins for centriole duplication have been identified. Two of them, SAS‐5 and SAS‐6, physically interact with each other and are codependent for their targeting to procentrioles. However, it remains unclear how these two proteins interact at the molecular level. Here, we demonstrate that the short SAS‐5 C‐terminal domain (residues 390–404) specifically binds to a narrow central region (residues 275–288) of the SAS‐6 coiled coil. This was supported by the crystal structure of the SAS‐6 coiled‐coil domain (CCD), which, together with mutagenesis studies, indicated that the association is mediated by synergistic hydrophobic and electrostatic interactions. The crystal structure also shows a periodic charge pattern along the SAS‐6 CCD, which gives rise to an anti‐parallel tetramer. Overall, our findings establish the molecular basis of the specific interaction between SAS‐5 and SAS‐6, and suggest that both proteins individually adopt an oligomeric conformation that is disrupted upon the formation of the hetero‐complex to facilitate the correct assembly of the nine‐fold symmetric centriole.     

Extreme C terminus of bacterial cytoskeletal protein FtsZ plays fundamental role in assembly independent of modulatory proteins

Bacterial cell division typically requires assembly of the cytoskeletal protein FtsZ into a ring (Z-ring) at the nascent division site that serves as a foundation for assembly of the division apparatus. High resolution imaging suggests that the Z-ring consists of short, single-stranded polymers held together by lateral interactions. Several proteins implicated in stabilizing the Z-ring enhance lateral interactions between FtsZ polymers in vitro. Here we report that residues at the C terminus of Bacillus subtilis FtsZ (C-terminal variable region (CTV)) are both necessary and sufficient for stimulating lateral interactions in vitro in the absence of modulatory proteins. Swapping the 6-residue CTV from B. subtilis FtsZ with the 4-residue CTV from Escherichia coli FtsZ completely abolished lateral interactions between chimeric B. subtilis FtsZ polymers. The E. coli FtsZ chimera readily formed higher order structures normally seen only in the presence of molecular crowding agents. CTV-mediated lateral interactions are important for the integrity of the Z-ring because B. subtilis cells expressing the B. subtilis FtsZ chimera had a low frequency of FtsZ ring formation and a high degree of filamentation relative to wild-type cells. Site-directed mutagenesis of the B. subtilis CTV suggests that electrostatic forces are an important determinant of lateral interaction potential.