Johannes D. Clausen

Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection.

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia‐containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule‐associated Chlamydia vesicles also collocated with tyrosine‐phosphorylated McCoy cell proteins. After migration, the Chlamydia‐containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule‐dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule‐dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR‐1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR‐1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis.

Ca2+-ATPases in non-failing and failing heart: evidence for a novel cardiac sarco/endoplasmic reticulum Ca2+-ATPase 2 isoform (SERCA2c).

We recently documented the expression of a novel human mRNA variant encoding a yet uncharacterized SERCA [SR (sarcoplasmic reticulum)/ER (endoplasmic reticulum) Ca2+-ATPase] protein, SERCA2c [Gélébart, Martin, Enouf and Papp (2003) Biochem. Biophys. Res. Commun. 303, 676-684]. In the present study, we have analysed the expression and functional characteristics of SERCA2c relative to SERCA2a and SERCA2b isoforms upon their stable heterologous expression in HEK-293 cells (human embryonic kidney 293 cells). All SERCA2 proteins induced an increased Ca2+ content in the ER of intact transfected cells. In microsomes prepared from transfected cells, SERCA2c showed a lower apparent affinity for cytosolic Ca2+ than SERCA2a and a catalytic turnover rate similar to SERCA2b. We further demonstrated the expression of the endogenous SERCA2c protein in protein lysates isolated from heart left ventricles using a newly generated SERCA2c-specific antibody. Relative to the known uniform distribution of SERCA2a and SERCA2b in cardiomyocytes of the left ventricle tissue, SERCA2c was only detected in a confined area of cardiomyocytes, in close proximity to the sarcolemma. This finding led us to explore the expression of the presently known cardiac Ca2+-ATPase isoforms in heart failure. Comparative expression of SERCAs and PMCAs (plasma-membrane Ca2+-ATPases) was performed in four nonfailing hearts and five failing hearts displaying mixed cardiomyopathy and idiopathic dilated cardiomyopathies. Relative to normal subjects, cardiomyopathic patients express more PMCAs than SERCA2 proteins. Interestingly, SERCA2c expression was significantly increased (166+/-26%) in one patient. Taken together, these results demonstrate the expression of the novel SERCA2c isoform in the heart and may point to a still unrecognized role of PMCAs in cardiomyopathies.

Purified E255L mutant SERCA1a and purified PFATP6 are sensitive to SERCA-type inhibitors but insensitive to artemisinins

The antimalarial drugs artemisinins have been described as inhibiting Ca2+-ATPase activity of PfATP6 (Plasmodium falciparum ATP6) after expression in Xenopus oocytes. Mutation of an amino acid residue in mammalian SERCA1 (Glu255) to the equivalent one predicted in PfATP6 (Leu) was reported to induce sensitivity to artemisinin in the oocyte system. However, in the present experiments, we found that artemisinin did not inhibit mammalian SERCA1a E255L either when expressed in COS cells or after purification of the mutant expressed in Saccharomyces cerevisiae. Moreover, we found that PfATP6 after expression and purification from S. cerevisiae was insensitive to artemisinin and significantly less sensitive to thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone than rabbit SERCA1 but retained higher sensitivity to cyclopiazonic acid, another type of SERCA1 inhibitor. Although mammalian SERCA and purified PfATP6 appear to have different pharmacological profiles, their insensitivity to artemisinins suggests that the mechanism of action of this class of drugs on the calcium metabolism in the intact cell is complex and cannot be ascribed to direct inhibition of PfATP6. Furthermore, the successful purification of PfATP6 affords the opportunity to develop new antimalarials by screening for inhibitors against PfATP6.

Mutational analysis of the conserved TGES loop of sarcoplasmic reticulum Ca2+-ATPase

Crystal structures have shown that the conserved TGES loop of the Ca2+-ATPase is isolated in the Ca2E1 state but becomes inserted in the catalytic site in E2 states. Here, we have examined the kinetics of the partial reaction steps of the transport cycle and the binding of the phosphoryl analogs BeF, AlF, MgF, and vanadate in mutants with alterations to the TGES residues. The mutations encompassed variation of size, polarity, and charge of the side chains. Differential effects on the Ca2E1P → E2P, E2P → E2, and E2 → Ca2E1 reactions and the binding of the phosphoryl analogs were observed. In the E183D mutant, the E2P → E2 dephosphorylation reaction proceeded at a rate as high as one-third that of the wild type, whereas it was very slow in the other Glu183 mutants, including E183Q, thus demonstrating the need for a negatively charged carboxylate group to catalyze dephosphorylation. By contrast, the Ca2E1P → E2P transition was accomplished at a reasonable rate with glutamine in place of Glu183, but not with aspartate, indicating that the length of the Glu183 side chain, in addition to its hydrogen bonding potential, is critical for Ca2E1P → E2P. This transition was also slowed in mutants with alteration to other TGES residues. The data provide functional evidence in support of the proposed role of Glu183 in activating the water molecule involved in the E2P → E2 dephosphorylation and suggest a direct participation of the side chains of the TGES loop in the control and facilitation of the insertion of the loop in the catalytic site. The interactions of the TGES loop furthermore seem to facilitate its disengagement from the catalytic site during the E2 → Ca2E1 transition.

Crystal Structure of D351A and P312A Mutant Forms of the Mammalian Sarcoplasmic Reticulum Ca2+-ATPase Reveals Key Events in Phosphorylation and Ca2+ Release

In recent years crystal structures of the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a), stabilized in various conformations with nucleotide and phosphate analogs, have been obtained. However, structural analysis of mutant forms would also be valuable to address key mechanistic aspects. We have worked out a procedure for affinity purification of SERCA1a heterologously expressed in yeast cells, producing sufficient amounts for crystallization and biophysical studies. We present here the crystal structures of two mutant forms, D351A and P312A, to address the issue whether the profound functional changes seen for these mutants are caused by major structural changes. We find that the structure of P312A with ADP and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{AlF}_{4}^{-}\) \end{document} bound (3.5-Å resolution) and D351A with AMPPCP or ATP bound (3.4- and 3.7-Å resolution, respectively) deviate only slightly from the complexes formed with that of wild-type ATPase. ATP affinity of the D351A mutant was very high, whereas the affinity for cytosolic Ca2+ was similar to that of the wild type. We conclude from an analysis of data that the extraordinary affinity of the D351A mutant for ATP is caused by the electrostatic effects of charge removal and not by a conformational change. P312A exhibits a profound slowing of the Ca2+-translocating Ca2E1P→E2P transition, which seems to be due to a stabilization of Ca2E1P rather than a destabilization of E2P. This can be accounted for by the strain that the Pro residue induces in the straight M4 helix of the wild type, which is removed upon the replacement of Pro312 with alanine in P312A.

SERCA mutant E309Q binds two Ca 2+ ions but adopts a catalytically incompetent conformation

The sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) couples ATP hydrolysis to transport of Ca2+. This directed energy transfer requires cross‐talk between the two Ca2+ sites and the phosphorylation site over 50 Å distance. We have addressed the mechano‐structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu309 contributes to Ca2+ coordination at site II, and a consensus has been that E309Q only binds Ca2+ at site I. The crystal structure of E309Q in the presence of Ca2+ and an ATP analogue, however, reveals two occupied Ca2+ sites of a non‐catalytic Ca2E1 state. Ca2+ is bound with micromolar affinity by both Ca2+ sites in E309Q, but without cooperativity. The Ca2+‐bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A‐domain, requiring a shift of transmembrane segment M1 into an ‘up and kinked position’. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca2+ site II.

ATP-binding Modes and Functionally Important Interdomain Bonds of Sarcoplasmic Reticulum Ca2+-ATPase Revealed by Mutation of Glycine 438, Glutamate 439, and Arginine 678

ATP binds to sarcoplasmic reticulum Ca2+-ATPase both in a phosphorylating (catalytic) mode and in a nonphosphorylating (modulatory) mode, the latter leading to acceleration of phosphoenzyme turnover (Ca2E1P → E2P and E2P → E2 reactions) and Ca2+ binding (E2 → Ca2E1). In some of the Ca2+-ATPase crystal structures, Arg678 and Glu439 seem to be involved in the binding of nucleotide or an associated Mg2+ ion. We have replaced Arg678, Glu439, and Gly438 with alanine to examine their importance for the enzyme cycle and the modulatory effects of ATP and MgATP. The results point to the key role of Arg678 in nucleotide binding and to the importance of interdomain bonds Glu439-Ser186 and Arg678-Asp203 in stabilizing the E2P and E2 intermediates, respectively. Mutation of Arg678 had conspicuous effects on ATP/MgATP binding to the E1 form and ADP binding to Ca2E1P, as well as ATP/MgATP binding in modulatory modes to E2P and E2, whereas the effects on ATP/MgATP acceleration of the Ca2E1P → E2P transition were small, suggesting that the nucleotide that accelerates Ca2E1P → E2P binds differently from that modulating the E2P → E2 and E2 → Ca2E1 reactions. Mutation of Glu439 hardly affected nucleotide binding to E1, Ca2E1P, and E2, but it led to disruption of the modulatory effect of ATP on E2P → E2 and acceleration of the latter reaction, indicating that ATP normally modulates E2P → E2 by interfering with the interaction between Glu439 and Ser186. Gly438 seems to be important for this interaction as well as for nucleotide binding, probably because of its role in formation of the helix containing Glu439 and Thr441.

ATP Binding Residues of Sarcoplasmic Reticulum Ca2+-ATPase

Abstract: ATP‐binding residues in the N and P domains of sarcoplasmic reticulum Ca‐ATPase have been investigated using mutagenesis in combination with a binding assay based on the photolabeling of Lys492 with [g‐32P] 2′,3′‐O‐(2,4,6 trinitrophenyl)‐8‐azido‐ATP and competition with nucleotide. In the N domain, mutations to several residues in conserved motifs, 438GEATE, 487FSRDRK, 515KGAPE, and 560RCLALA produce nucleotide‐binding defects. Key residues include Thr441, Glu442, Phe487, Arg489, Lys492, Lys515, Arg560, and Leu562. In the absence of Mg2+, Arg489, Lys492, and Arg560 are most important, whereas in its presence Thr441 and Glu442 also play a crucial role. In the P domain, Asp351 is striking for its strong electrostatic repulsion of the γ‐phosphate, especially in the presence of Mg2+. Lys352 is a key residue, and Asp627 and Lys684 must come close to the nucleotide. Thr353, Asn359, Asp601, and Asp703 interact only in the presence of Mg2+. Asn706 and Asp707 are unimportant for nucleotide binding. The results identify several ATP binding residues in the N and P domains and suggest that Mg2+ changes the nucleotide/protein interaction in both. Models of bound ATP and MgATP are presented.

Modulatory ATP binding affinity in intermediate states of E2P dephosphorylation of sarcoplasmic reticulum Ca2+-ATPase

The mechanism of ATP modulation of E2P dephosphorylation of sarcoplasmic reticulum Ca2+-ATPase wild type and mutant forms was examined in nucleotide binding studies of states analogous to the various intermediates of the dephosphorylation reaction, obtained by binding of metal fluorides, vanadate, or thapsigargin. Wild type Ca2+-ATPase displays an ATP affinity of 4 μm for the E2P ground state analog, 1 μm for the E2P transition state and product state analogs, and 11 μm for the E2 dephosphoenzyme. Hence, ATP binding stabilizes the transition and product states relative to the ground state, thereby explaining the accelerating effect of ATP on dephosphorylation. Replacement of Phe487 (N-domain) with serine, Arg560 (N-domain) with leucine, or Arg174 (A-domain) with alanine or glutamate reduces ATP affinity in all E2/E2P intermediate states. Alanine substitution of Ile188 (A-domain) increases the ATP affinity, although ATP acceleration of dephosphorylation is disrupted, thus indicating that the critical role of Ile188 in ATP modulation is mechanistically based rather than being associated with the binding of nucleotide. Mutants with alanine replacement of Lys205 (A-domain) or Glu439 (N-domain) exhibit an anomalous inhibition by ATP of E2P dephosphorylation, due to ATP binding increasing the stability of the E2P ground state relative to the transition state. The ATP affinity of Ca2E2P, stabilized by inserting four glycines in the A-M1 linker, is similar to that of the E2P ground state, but the Ca2+-free E1 state of this mutant exhibits 3 orders of magnitude reduction of ATP affinity.

Asparagine 706 and glutamate 183 at the catalytic site of sarcoplasmic reticulum Ca2+-ATPase play critical but distinct roles in E2 states.

Mutants with alteration to Asn706 of the highly conserved 701TGDGVND707 motif in domain P of sarcoplasmic reticulum Ca2+-ATPase were analyzed for changes in transport cycle kinetics and binding of the inhibitors vanadate, BeF, AlF, and MgF. The fluorides likely mimic the phosphoryl group/Pi in the respective ground, transition, and product states of phosphoenzyme hydrolysis (Danko, S., Yamasaki, K., Daiho, T., and Suzuki, H. (2004) J. Biol. Chem. 279, 14991–14998). Binding of BeF, AlF, and MgF was also studied for mutant Glu183 → Ala, where the glutamate of the 181TGES184 motif in domain A is replaced. Mutations of Asn706 and Glu183 have in common that they dramatically impede the function of the enzyme in E2 states, but have little effect in E1. Contrary to the Glu183 mutant, in which E2P slowly accumulates (Clausen, J. D., Vilsen, B., McIntosh, D. B., Einholm, A. P., and Andersen, J. P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2776–2781), E2P formation was not detectable with the Asn706 mutants. Differential sensitivities of the mutants to inhibition by AlF, MgF, and BeF made it possible to distinguish different roles of Asn706 and Glu183. Hence, Asn706 is less important than Glu183 for gaining the transition state during E2P hydrolysis but plays critical roles in stabilization of E2P ground and E2·Pi product states and in the major conformational changes associated with the Ca2E1P → E2P and E2 → Ca2E1 transitions, which seem to be facilitated by interaction of Asn706 with domain A.