The assembly of the PsaD subunit into the membranal photosystem I complex occurs via an exchange mechanism.

PsaD is a peripheral stromal-facing subunit of photosystem I (PSI), a multisubunit complex of the thylakoid membranes. PsaD plays a major role in both the function and assembly of PSI. Past studies with radiolabeled PsaD indicated that PsaD is able to assemble in vitro specifically into the PSI complex. To unravel the mechanism by which this assembly takes place, the following steps were taken. (i) Mature PsaD of spinach and PsaD of the prokaryotic caynobacterium Mastigocladus laminosus, both bearing a six-histidine tag at their C-termini, were overexpressed in Escherichia coli and purified to homogeneity. (ii) The purified recombinant protein was introduced into the isolated PSI complex. (iii) Following incubation, the PsaD that assembled into PSI was separated from the nonassembled PsaD by a sucrose gradient. Differential Western blot analysis was used to determine whether the native and the recombinant PsaD were present as free or assembled proteins of the PSI complex. Antibodies that can recognize only the recombinant PsaD (anti-his) or both the native and recombinant PsaD (anti-PsaD) were used. The findings indicated that an exchange mechanism enables the assembly of a newly introduced PsaD into PSI. The latter replaces the PsaD subunit that is present in situ within the complex. In vivo studies that followed the assembly of PsaD in Chlamydomonas reinhardtii cells supported this in vitro-characterized exchange mechanism. In C. reinhardtii, in the absence of synthesis and assembly of new PSI complexes, newly synthesized PsaD assembled into pre-existing PSI complexes.     

Interaction of the soluble recombinant PsaD subunit of spinach photosystem I with ferredoxin I.

Photosystem I of higher plants functions in photosynthesis as a light-driven oxidoreductase producing reduced ferredoxin. Its peripheral subunit PsaD has been identified as the docking site for ferredoxin I. With the aim of elucidating the structure-function relationship and the role of this subunit, a recombinant form of the spinach protein was produced by heterologous expression in Escherichia coli. The PsaD protein was synthesized in soluble form and purified to homogeneity. The interaction of the PsaD subunit with ferredoxin I was investigated using three different approaches: chemical cross-linking between the two purified proteins in solution, affinity chromatography of the PsaD subunit on a ferredoxin-coupled resin, and titration with ferredoxin of the protein fluorescence of the subunit. All these studies indicated that the isolated PsaD in solution has a definite conformation and maintains the ability to bind ferredoxin I with high affinity and specificity. The Kd value of the complex of PsaD and ferredoxin is in the nanomolar range, which is consistent with reported Km values for ferredoxin I photoreduction by thylakoid membranes. The ionic strength dependence of the K(d) suggests that the protein-protein interaction is at least partially electrostatic in nature. Nevertheless, none of the glutamate residues of the acidic cluster of residues 92-94 of ferredoxin I, which have been reported to be involved in the interaction with the subunit, seems to be essential for PsaD binding, as borne out by experiments using ferredoxin I mutants in positions 92-94.     

An insight into the assembly and organization of photosystem I complex in the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus

The present study characterizes the assembly and organization of Photosystem I (PSI) complex, and its individual subunits into the thylakoid membranes of the thermophilic cyanobacterium, Mastigocladus laminosus. PSI is a multiprotein complex that contains peripheral as well as integral subunits. Hence, it serves as a suitable model system for understanding the formation and organization of membrane protein complexes. In the present study, two peripheral cytosol facing subunits of PSI, namely, PsaD and PsaE were overexpressed in E. coli and used for assembly studies. The gene encoding PsaK, an integral membrane spanning subunit of PSI, was cloned and the deduced amino acid sequence revealed PsaK to have two transmembrane alpha-helices. The characterization of the in vitro assembly of the peripheral subunits, PsaD and PsaE, as well as of the integral subunit, PsaK, was performed by incubating each subunit with thylakoids isolated from Mastigocladus laminosus. All three subunits studied were found to assemble into the thylakoids in a spontaneous mechanism, showing no requirement for cytosolic factors or NTP's (nucleotide 5'-triphosphate). Nevertheless, further characterization of the assembly of PsaK revealed its membrane integration to be most efficient at 55 degrees C. The associations and protein-protein interactions between different subunits within the assembled PSI complex were directly quantified by measurements performed using the BIACORE technology. The preliminary results indicated the existence of specific interaction between PsaD and PsaE, and revealed a very high binding affinity between PsaD and the PSI electron acceptor ferridoxin (Kd = 5.8 x 10(-11) M). PsaE has exhibited a much lower binding affinity for ferridoxin (Kd = 3.1 x 10(-5) M), thereby supporting the possibility of PsaE being one of the subunits responsible for the dissociation of ferridoxin from the PSI complex.     

Molecular characterization and overexpression of the petF gene from Synechococcus elongatus: Evidence for a second site of electrostatic interaction between ferredoxin and the PS I-D subunit

The petF gene from the cyanobacterium Synechococcus elongatus was isolated using the same gene from Synechocystis sp. PCC 6803 as a heterologous probe. The deduced primary sequence of the isolated single copy petF gene is identical to the primary sequence determined from the protein. Wild-type ferredoxin and a E93-95/Q93-95 mutant were overexpressed in E. coli and purified. Both types of ferredoxins are photoreduced by Photosystem I and can be cross-linked to the PsaD subunit of PS I, although with reduced affinity in case of the E93-95/Q93-95 mutant. These data indicate that the acidic patch of amino acids Glu94-95 of ferredoxin is most likely neither essential for the interaction of ferredoxin with PS I nor the only site of electrostatic contact with the PS I-D subunit. In contrast, NADP⁺photoreduction assays show drastically reduced rates in the presence of the E93-95/Q93-95 mutant ferredoxin, indicating that these residues play a crucial role in the interaction of ferredoxin with ferredoxin-NADP⁺reductase.     

The carboxyl-terminal region of the spinach PsaD subunit contains information for its specific assembly into plant thylakoids

The assembly of the multi-subunit membrane-protein Photosystem I (PS I) complex involves incorporation of peripheral proteins into the complex. Here we studied assembly of the PsaD subunit of the cyanobacterial and plant PS I into the thylakoid membranes. We generated partial and chimeric psaD genes from which labeled proteins were synthesized in vitro. Assembly of these proteins into the cyanobacterial or plant thylakoids was assayed. The deletion of leader sequence and N-terminal extension of spinach prePsaD did not inhibit its assembly into spinach or cyanobacterial thylakoids. Addition of these sequences to the cyanobacterial PsaD did not enable it to assemble into plant thylakoids. Moreover, these additions significantly decreased the ability of the chimeric proteins to assemble into cyanobacterial thylakoids. In contrast, when the carboxyl-terminal half of cyanobacterial PsaD was replaced by the corresponding region of the spinach PsaD, the chimeric protein could assemble into both spinach and cyanobacterial thylakoids. Therefore, information in the carboxyl-terminal region of spinach PsaD is crucial for its assembly into plant thylakoids.Abbreviation prePsaD precursor of the PsaD subunit of PS I     

Identification of the Escherichia coli enzyme I binding site in histidine-containing protein, HPr, by the effects of mutagenesis.

The structure of the N-terminal domain of enzyme I complexed with histidine-containing protein (HPr) has been described by multi-dimensional NMR. Residues in HPr involved in binding were identified by intermolecular nuclear Overhauser effects (Garrett et al. 1999). Most of these residues have been mutated, and the effect of these changes on binding has been assessed by enzyme I kinetic measurement. Changes to Thr16, Arg17, Lys24, Lys27, Ser46, Leu47, Lys49, Gln51, and Thr56 result in increases to the HPr Km of enzyme I, which would be compatible with changes in binding. Except for mutations to His15 and Arg17, very little or no change in Vmax was found. Alanine replacements for Gln21, Thr52, and Leu55 have no effect. The mutation Lys40Ala also affects HPr Km of enzyme I; residue 40 is contiguous with the enzyme I binding site in HPr and was not identified by NMR. The mutations leading to a reduction in the size of the side chain (Thr16Ala, Arg17Gly, Lys24Ala, Lys27Ala, and Lys49Gly) caused relatively large increases in Km (>5-fold) indicating these residues have more significant roles in binding to enzyme I. Acidic replacement at Ser46 caused very large increases (>100-fold), while Gln51Glu gave a 3-fold increase in Km. While these results essentially concur with the identification of residues by the NMR experiments, the apparent importance of individual residues as determined by mutation and kinetic measurement does not necessarily correspond with the number of contacts derived from observed intermolecular nuclear Overhauser effects.     

Molecular characterization of an extended binding site for coagulation factor Va in the positive exosite of activated protein C

The anticoagulant human plasma serine protease, activated protein C (APC), inhibits blood coagulation by specific inactivation of the coagulation cofactors factor Va (FVa) and factor VIIIa. Site-directed mutagenesis of residues in three surface loops of a positive exosite located on APC was used to identify residues that play a significant role in binding to FVa. Eighteen different residues were mutated to alanine singly, in pairs, or in triple mutation combinations. Mutant APC proteins were purified and characterized for their inactivation of FVa. Three APC residues were identified that provide major contributions to FVa interactions: Lys(193), Arg(229), and Arg(230). In addition, four residues made significant minor contributions to FVa interactions: Lys(191), Lys(192), Asp(214), and Glu(215). All of these residues primarily contribute to APC cleavage at Arg(506) in FVa and play a small role in the interaction of APC with the Arg(306) cleavage site. In conjunction with previously published work, these results define an extensive FVa binding site in the positive exosite of APC that is primarily involved in binding and cleaving at Arg(506) on FVa.     

Roles of N-terminal region residues Lys11, Arg13, and Arg24 of antithrombin in heparin recognition and in promotion and stabilization of the heparin-induced conformational change

The N-terminal region residues, Lys11, Arg13, and Arg24, of the plasma coagulation inhibitor, antithrombin, have been implicated in binding of the anticoagulant polysaccharide, heparin, from the identification of natural mutants with impaired heparin binding or by the X-ray structure of a complex of the inhibitor with a high-affinity heparin pentasaccharide. Mutations of Lys11 or Arg24 to Ala in this work each reduced the affinity for the pentasaccharide approximately 40-fold, whereas mutation of Arg13 to Ala led to a decrease of only approximately 7-fold. All three substitutions resulted in the loss of one ionic interaction with the pentasaccharide and those of Lys11 or Arg24 also in 3-5-fold losses in affinity of nonionic interactions. Only the mutation of Lys11 affected the initial, weak interaction step of pentasaccharide binding, decreasing the affinity of this step approximately 2-fold. The mutations of Lys11 and Arg13 moderately, 2-7-fold, altered both rate constants of the second, conformational change step, whereas the substitution of Arg24 appreciably, approximately 25-fold, reduced the reverse rate constant of this step. The N-terminal region of antithrombin is thus critical for high-affinity heparin binding, Lys11 and Arg24 being responsible for maintaining appreciable and comparable binding energy, whereas Arg13 is less important. Lys11 is the only one of the three residues that is involved in the initial recognition step, whereas all three residues participate in the conformational change step. Lys11 and Arg13 presumably bind directly to the heparin pentasaccharide by ionic, and in the case of Lys11, also nonionic interactions. However, the role of Arg24 most likely is indirect, to stabilize the heparin-induced P-helix by interacting intramolecularly with Glu113 and Asp117, thereby positioning the crucial Lys114 residue for optimal ionic and nonionic interactions with the pentasaccharide. Together, these findings show that N-terminal residues of antithrombin make markedly different contributions to the energetics and dynamics of binding of the pentasaccharide ligand to the native and activated conformational states of the inhibitor that could not have been predicted from the X-ray structure.     

Thrombin interacts with thrombomodulin, protein C, and thrombin-activatable fibrinolysis inhibitor via specific and distinct domains.

A collection of 56 purified thrombin mutants, in which 76 charged or polar surface residues on thrombin were mutated to alanine, was used to identify key residues mediating the interactions of thrombin with thrombomodulin (TM), protein C, and thrombin-activatable fibrinolysis inhibitor (TAFI). Comparison of protein C activation in the presence and absence of TM identified 11 residues mediating the thrombin-TM interaction (Lys(21), Gln(24), Arg(62), Lys(65), His(66), Arg(68), Thr(69), Tyr(71), Arg(73), Lys(77), Lys(106)). Three mutants (E25A, D51A, R89A/R93A/E94A) were found to have decreased ability to activate TAFI yet retained normal protein C activation, whereas three other mutants (R178A/R180A/D183A, E229A, R233A) had decreased ability to activate protein C but maintained normal TAFI activation. One mutant (W50A) displayed decreased activation of both substrates. Mapping of these functional residues on thrombin revealed that the 11 residues mediating the thrombin-TM interaction are all located in exosite I. Residues important in TAFI activation are located above the active-site cleft, whereas residues involved in protein C are located below the active-site cleft. In contrast to the extensive overlap of residues mediating TM binding and fibrinogen clotting, these data show that distinct domains in thrombin mediate its interactions with TM, protein C, and TAFI. These studies demonstrate that selective enzymatic properties of thrombin can be dissociated by site-directed mutagenesis.     

Site-Directed Mutagenesis of Cytochrome P450scc (CYP11A1). Effect of Lysine Residue Substitution on Its Structural and Functional Properties

Our previous chemical modification and cross-linking studies identified some positively charged amino acid residues of cytochrome P450scc that may be important for its interaction with adrenodoxin and for its functional activity. The present study was undertaken to further evaluate the role of these residues in the interaction of cytochrome P450scc with adrenodoxin using site-directed mutagenesis. Six cytochrome P450scc mutants containing replacements of the surface-exposed positively charged residues (Lys103Gln, Lys110Gln, Lys145Gln, Lys394Gln, Lys403Gln, and Lys405Gln) were expressed in E. coli cells, purified as a substrate-bound high-spin form, and characterized as compared to the wild-type protein. The replacement of the surface Lys residues does not dramatically change the protein folding or the heme pocket environment as judged from limited proteolysis and spectral studies of the cytochrome P450 mutants. The replacement of Lys in the N-terminal sequence of P450scc does not dramatically affect the activity of the heme protein. However, mutant Lys405Gln revealed rather dramatic loss of cholesterol side-chain cleavage activity, efficiency of enzymatic reduction in a reconstituted system, and apparent dissociation constant for adrenodoxin binding. The present results, together with previous findings, suggest that the changes in functional activity of mutant Lys405Gln may reflect the direct participation of this amino acid residue in the electrostatic interaction of cytochrome P450scc with its physiological partner, adrenodoxin.     

Identification of a novel prohormone sorting signal-binding site on carboxypeptidase E, a regulated secretory pathway-sorting receptor

Sorting of the prohormone POMC to the regulated secretory pathway necessitates the binding of a sorting signal to a sorting receptor, identified as membrane carboxypeptidase E (CPE). The sorting signal, located at the N terminus of POMC consists of two acidic (Asp10, Glu14) and two hydrophobic (Leu11, Leu18) residues exposed on the surface of an amphipathic loop. In this study, molecular modeling of CPE predicted that the acidic residues in the POMC-sorting signal bind specifically to two basic residues, Arg255 and Lys260, present in a loop unique to CPE, compared with other carboxypeptidases. To test the model, these two residues on CPE were mutated to Ser or Ala, followed by baculovirus expression of the mutant CPEs in Sf9 cells. Sf9 cell membranes containing CPE mutants with either Arg255 or Lys260, or both residues substituted, showed no binding of [125I]N-POMC1-26 (which contains the POMC-sorting signal motif), proinsulin, or proenkephalin. In contrast, substitution of an Arg147 to Ala147 at a substrate-binding site, Arg259 to Ala259 and Ser202 to Pro202, in CPE did not affect the level of [125I]N-POMC1-26 binding when compared with-wild type CPE. Furthermore, mutation of the POMC-sorting signal motif (Asp10, Leu11, Glu14, Leu18) eliminated binding to wild-type CPE. These results indicate that the sorting signal of POMC, proinsulin, and proenkephalin specifically interacts with Arg255 and Lys260 at a novel binding site, independent of the active site on CPE.     

Structural and functional characterization of the first intracellular loop of human thromboxane A2 receptor

The conformation of a constrained peptide mimicking the putative first intracellular domain (iLP1) of thromboxane A(2) receptor (TP) was determined by (1)H 2D NMR spectroscopy. Through completed assignments of TOCSY, DQF-COSY, and NOESY spectra, a NMR structure of the peptide showed a beta-turn in residues 56-59 and a short helical structure in the residues 63-66. It suggests that residues 63-66 may be part of the second transmembrane domain (TM), and that Arg60, in an exposed position on the outer surface of the loop, may be involved in signaling through charge contact with Gq protein. The sequence alignment of Lys residue in the same position of other prostanoid receptors mediates different G protein couplings, suggesting that the chemical properties of Arg and Lys may also affect the receptor signaling activity. These hypotheses were supported by mutagenesis studies, in which the mutant of Arg60Leu completely lost activity in increasing intracellular calcium level through Gq coupling, and the mutant of Arg60Lys retained only about 35% signaling activity. The difference between the side chain functions of Lys and Arg in effecting the signaling was discussed.     

Identification of contact residues and definition of the CAR-binding site of adenovirus type 5 fiber protein

The binding of adenovirus (Ad) fiber knob to its cellular receptor, the coxsackievirus and Ad receptor (CAR), promotes virus attachment to cells and is a major determinant of Ad tropism. Analysis of the kinetics of binding of Ad type 5 (Ad5) fiber knob to the soluble extracellular domains of CAR together (sCAR) and each immunoglobulin (Ig) domain (IgV and IgC2) independently by surface plasmon resonance demonstrated that the IgV domain is necessary and sufficient for binding, and no additional membrane components are required to confer high-affinity binding to Ad5 fiber knob. Four Ad5 fiber knob mutations, Ser408Glu and Pro409Lys in the AB loop, Tyr477Ala in the DG loop, and Leu485Lys in beta strand F, effectively abolished high-affinity binding to CAR, while Ala406Lys and Arg412Asp in the AB loop and Arg481Glu in beta strand E significantly reduced the level of binding. Circular dichroism spectroscopy showed that these mutations do not disorder the secondary structure of the protein, implicating Ser408, Pro409, Tyr477, and Leu485 as contact residues, with Ala406, Arg412, and Arg481 being peripherally or indirectly involved in CAR binding. The critical residues have exposed side chains that form a patch on the surface, which thus defines the high-affinity interface for CAR. Additional site-directed mutagenesis of Ad5 fiber knob suggests that the binding site does not extend to the adjacent subunit or toward the edge of the R sheet. These findings have implications for our understanding of the biology of Ad infection, the development of novel Ad vectors for targeted gene therapy, and the construction of peptide inhibitors of Ad infection.     

Characterization of DNA-binding activity in the N-terminal domain of the DNA methyltransferase Dnmt3a

The Dnmt3a gene, which encodes de novo-type DNA methyltransferase, encodes two isoforms, full-length Dnmt3a and Dnmt3a2, which lacks the N-terminal 219 amino acid residues. We found that Dnmt3a showed higher DNA-binding and DNA-methylation activities than Dnmt3a2. The N-terminal sequence from residues 1 to 211 was able to bind to DNA, but could not distinguish methylated and unmethylated CpG. Its binding to DNA was inhibited by a major groove binder. Four basic amino acid residues, Lys51, Lys53, Arg177 and Arg179, in the N-terminal region were crucial for the DNA-binding activity. The ectopically expressed N-terminal sequence (residues 1-211) was localized in nuclei, whereas that harbouring mutations at the four basic amino acid residues was also detected in the cytoplasm. The DNA-methylation activity of Dnmt3a with the mutations was suppressed under physiological salt conditions, which is similar that of Dnmt3a2. In addition, ectopically expressed Dnmt3a with mutations, as well as Dnmt3a2, could not be retained efficiently in nuclei on salt extraction. We conclude that the DNA-binding activity of the N-terminal domain contributes to the DNA-methyltransferase activity via anchoring of the whole molecule to DNA under physiological salt conditions.     

A basic residue, Lys 782, composes part of the ATP-binding site on the epidermal growth factor receptor tyrosine kinase

To identify amino acids specific for tyrosine kinase activity, the role of several conserved basic residues in kinase function was tested. Modeling of the epidermal growth factor receptor tyrosine kinase domain based on the crystal structure of cyclic AMP-dependent protein kinase and insulin receptor revealed several basic residues present on the surface of epidermal growth factor receptor. Using the molecular modeling program, GRASP, the basic residues Arg 779, Lys 782, and Lys 855 were shown to provide an area of positive charge to the surface of the molecule. To deduce the role of these residues in ATP and substrate binding, site-directed mutants were prepared and kinetic constants were measured. Mutation of Lys 855 to Ala destabilized the enzyme and caused partial inactivation. Mutation of either Arg 779 or Lys 782 had little effect on the Km value for peptide substrate. However, alteration of Lys 782 increased the Km value for ATP 28-fold, indicating a role for Lys 782 in binding ATP. Because residues similar to Lys 782 in the sequences of mitogen-activated protein kinase and insulin receptor make contact with a ribose hydroxyl of ATP, it is proposed that Lys 782 may be one of the residues composing the ribose-binding site of epidermal growth factor receptor.     

Structural and mechanistic investigation of 3-deoxy-D-manno-octulosonate-8-phosphate synthase by solid-state REDOR NMR

15N?(31)P? REDOR NMR experiments were applied to lyophilized binary complexes of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), with each of its natural substrates, phosphoenolpyruvate (PEP) and arabinose-5-phsophate (A5P), and with a mechanism-based inhibitor (K(i) = 0.4 microM), directly characterizing the active site basic residues involved in the binding of their carboxylate and phosphate moieties. KDO8PS was labeled uniformly with (15)N or [eta-(15)N(2)]Arg, and the ligands were selectively labeled with (13)C and (15)N. The NMR data established that PEP is bound by KDO8PS via a preserved set of structurally rigid and chemically unique Arg and Lys residues, with 5 A (upper limit) between epsilon-(15)N of this Lys and (31)P of PEP. A5P is bound in its cyclic forms to KDO8PS via a different set of Lys and Arg residues. The two sets arise from adjacent subsites that are capable of independent and sufficiently strong binding. The inhibitor is best characterized as an A5P-based substrate analogue inhibitor of KDO8PS. Five mutants in which highly conserved arginines were replaced with alanines were prepared and kinetically characterized. Our solid-state NMR observations complement the crystallographic structure of KDO8PS, and in combination with the mutagenesis results enable tentative assignment of the NMR-identified active site residues. Lys-138 and Arg-168 located at the most recessed part of the active site cavity are the chemically distinct and structurally rigid residues that bind PEP phosphate; R168A resulted in 0.1% of wild-type activity. Arg-63, exposed at the opening of the active site barrel, is the flexible residue with a generic chemical shift that binds A5P; R63A resulted in complete deactivation. The mechanistic implications of our results are discussed.     

Identification of Photosystem I components from a glaucocystophyte,Cyanophora paradoxa: The PsaD protein has an N-terminal stretch homologous to higher plants

Thylakoid membranes and Photosystem I (PS I) complexes were isolated from a glaucocystophyte, Cyanophora paradoxa, which is thought to have the most primitive ‘plastids’, and the proteins related to PS I were examined. The intrinsic light-harvesting chlorophyll protein complexes of PS I (LHC I) were not detected by an immunological method. The PS I complexes consisted of at least eight low-molecular-mass proteins in addition to PS I reaction center proteins. The N-terminal sequence of the PsaD protein has higher homology to that of Chlamydomonas reinhardtii and land plants, than to that of other algae or cyanobacteria. On the other hand, the PsaL sequence has the highest homology to those of cyanobacteria. Taking into account the other sequences of PS I components whose genes are encoded in the cyanelle genome, and the fact that LHC I is not detected, it is concluded that PS I of C. paradoxa has chimeric characteristics of both ‘green’ lineages and cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.