Multisubunit Membrane Protein Complex Research Articles

Exploring the energetics of water permeation in photosystem II by multiple steered molecular dynamics simulations

The Mn4Ca cluster of the oxygen-evolving complex (OEC) of photosynthesis catalyzes the light-driven splitting of water into molecular oxygen, protons, and electrons. The OEC is buried within photosystem II (PSII), a multisubunit integral membrane protein complex, and water must find its way to the Mn4Ca cluster by moving through protein. Molecular dynamics simulations were used to determine the energetic barriers for water permeation though PSII extrinsic proteins. Potentials of mean force (PMFs) for water were derived by using the technique of multiple steered molecular dynamics (MSMD). Calculation of free energy profiles for water permeation allowed us to characterize previously identified water channels, and discover new pathways for water movement toward the Mn4Ca cluster. Our results identify the main constriction sites in these pathways which may serve as selectivity filters that restrict both the access of solutes detrimental to the water oxidation reaction and loss of Ca2+ and Cl− from the active site.

Low-resolution structure determination of Na+-translocating NADH:ubiquinone oxidoreductase fromVibrio choleraebyab initiophasing and electron microscopy

A low-resolution structure of the Na(+)-translocating NADH:ubiquinone oxidoreductase from the human pathogen Vibrio cholerae was determined by ab initio phasing and independently confirmed by electron microscopy. This multi-subunit membrane-protein complex (molecular weight 210 kDa) generates an Na(+) gradient that is essential for substrate uptake, motility, pathogenicity and efflux of antibiotics. The obtained 16 Å resolution electron density-map revealed an asymmetric particle with a central region of low electron density and a putative detergent region, and allowed the identification of the transmembrane regions of the complex.

Role of Novel Dimeric Photosystem II (PSII)-Psb27 Protein Complex in PSII Repair

The multisubunit membrane protein complex Photosystem II (PSII) catalyzes one of the key reactions in photosynthesis: the light-driven oxidation of water. Here, we focus on the role of the Psb27 assembly factor, which is involved in biogenesis and repair after light-induced damage of the complex. We show that Psb27 is essential for the survival of cyanobacterial cells grown under stress conditions. The combination of cold stress (30 °C) and high light stress (1000 μmol of photons × m(-2) × s(-1)) led to complete inhibition of growth in a Δpsb27 mutant strain of the thermophilic cyanobacterium Thermosynechococcus elongatus, whereas wild-type cells continued to grow. Moreover, Psb27-containing PSII complexes became the predominant PSII species in preparations from wild-type cells grown under cold stress. Two different PSII-Psb27 complexes were isolated and characterized in this study. The first complex represents the known monomeric PSII-Psb27 species, which is involved in the assembly of PSII. Additionally, a novel dimeric PSII-Psb27 complex could be allocated in the repair cycle, i.e. in processes after inactivation of PSII, by (15)N pulse-label experiments followed by mass spectrometry analysis. Comparison with the corresponding PSII species from Δpsb27 mutant cells showed that Psb27 prevented the release of manganese from the previously inactivated complex. These results indicate a more complex role of the Psb27 protein within the life cycle of PSII, especially under stress conditions.

The structural basis for intramembrane assembly of an activating immunoreceptor complex

Many receptors that activate cells of the immune system are multisubunit membrane protein complexes in which ligand recognition and signaling functions are contributed by separate protein modules. Receptors and signaling subunits assemble through contacts among basic and acidic residues in their transmembrane domains to form the functional complexes. Here we report the nuclear magnetic resonance (NMR) structure of the membrane-embedded, heterotrimeric assembly formed by association of the DAP12 signaling module with the natural killer (NK) cell-activating receptor NKG2C. The main intramembrane contact site is formed by a complex electrostatic network involving five hydrophilic transmembrane residues. Functional mutagenesis demonstrated that similar polar intramembrane motifs are also important for assembly of the NK cell-activating NKG2D-DAP10 complex and the T cell antigen receptor (TCR)-invariant signaling protein CD3 complex. This structural motif therefore lies at the core of the molecular organization of many activating immunoreceptors.

Cooperation of LPA3 and LPA2 is essential for photosystem II assembly in Arabidopsis.

Photosystem II (PSII) is a multisubunit membrane protein complex that is assembled in a sequence of steps. However, the molecular mechanisms responsible for the assembly of the individual subunits into functional PSII complexes are still largely unknown. Here, we report the identification of a chloroplast protein, Low PSII Accumulation3 (LPA3), which is required for the assembly of the CP43 subunit in PSII complexes in Arabidopsis (Arabidopsis thaliana). LPA3 interacts with LPA2, a previously identified PSII CP43 assembly factor, and a double mutation of LPA2 and LPA3 is more deleterious for assembly than either single mutation, resulting in a seedling-lethal phenotype. Our results indicate that LPA3 and LPA2 have overlapping functions in assisting CP43 assembly and that cooperation between LPA2 and LPA3 is essential for PSII assembly. In addition, we provide evidence that LPA2 and LPA3 interact with Albino3 (Alb3), which is essential for thylakoid protein biogenesis. Thus, the function of Alb3 in some PSII assembly processes is probably mediated through interactions with LPA2 and LPA3.

The Assembly of a Multisubunit Photosynthetic Membrane Protein Complex: A Site-Specific Spin Labeling EPR Spectroscopic Study of the PsaC Subunit in Photosystem I

The assembly of the PsaC subunit in the photosystem I (PS I) complex was studied using site-specific spin labeling electron paramagnetic resonance (EPR) spectroscopic techniques. The binding was monitored from the perspective of a reporter spin label attached to either the native C34(C) or the engineered C75(C) residue of wild-type PsaC (PsaC(WT)). Three distinct stages of PsaC assembly were analyzed: unbound PsaC, the P(700)-F(X)/PsaC complex, and the P(700)-F(X)/PsaC/PsaD complex. The changes in the EPR spectral line shape and the rotational correlation time of the spin label when PsaC(WT) binds to the PS I core are consistent with the conformational changes that are expected to occur during the assembly process. The addition of the PsaD subunit to the P(700)-F(X)/PsaC(WT-C34) complex induces further EPR spectral changes, which indicate that the presence of PsaD affects the orientation of the PsaC subunit on the PS I core. The binding of several PsaC variants, each lacking one or more key binding contacts with the PsaA/PsaB heterodimer, was monitored using a reporter spin label at C34(C). Our results indicate that the absence of the PsaC-PsaA/PsaB binding contacts causes PsaC to bind in an altered configuration on the PS I core. In particular, the removal of the entire C-terminus (PsaC(C-term)) causes PsaC to dock in a significantly different orientation when compared to the wild-type protein, as indicated by the EPR spectrum of the P(700)-F(X)/PsaC(C-term-C34) complex. Because the PsaC(C-term) variant retains only the symmetric network of PsaC-PsaA/PsaB ionic contacts, the altered EPR spectrum could, in principle, reflect a fraction of reaction centers that contain PsaC bound in the 180 degrees-rotated, C(2)-symmetry-related configuration. The results of this study are used to provide a comprehensive, stepwise mechanism for the binding of PsaC on the PS I core.

Tracking the Flow of Water through Photosystem II Using Molecular Dynamics and Streamline Tracing

The CaMn(4) cluster of the oxygen-evolving complex (OEC) of photosynthesis catalyzes the light-driven splitting of water into molecular oxygen, protons, and electrons. The OEC is buried within photosystem II (PSII), a multisubunit integral membrane protein complex, and water must find its way to the CaMn(4) cluster by moving through protein. Channels for water entrance, and proton and oxygen exit, have previously been proposed following the analysis of cavities found within X-ray structures of PSII. However, these analyses do not account for the dynamic motion of proteins and cannot track the movement of water within PSII. To study water dynamics in PSII, we performed molecular dynamics simulations and developed a novel approach for the visualization of water diffusion within protein based on a streamline tracing algorithm used in fluid dynamics and diffusion tensor imaging. We identified a system of branching pathways of water diffusion in PSII leading to the OEC that connect to a number of distinct entrance points on the lumenal surface. We observed transient changes in the connections between channels and entrance points that served to moderate both the flow of water near the OEC and the exchange of water inside and outside of the protein. Water flow was significantly altered in simulations lacking the OEC which were characterized by a simpler and wider channel with only two openings, consistent with the creation of an ion channel that allows entry of Mn(2+), Ca(2+), and Cl(-) as required for construction of the CaMn(4) cluster.

Comparative Structural Biology of Eubacterial and Archaeal Oligosaccharyltransferases

Oligosaccharyltransferase (OST) catalyzes the transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. In the bacterium Campylobacter jejuni, a single-subunit membrane protein, PglB, catalyzes N-glycosylation. We report the 2.8 A resolution crystal structure of the C-terminal globular domain of PglB and its comparison with the previously determined structure from the archaeon Pyrococcus AglB. The two distantly related oligosaccharyltransferases share unexpected structural similarity beyond that expected from the sequence comparison. The common architecture of the putative catalytic sites revealed a new catalytic motif in PglB. Site-directed mutagenesis analyses confirmed the contribution of this motif to the catalytic function. Bacterial PglB and archaeal AglB constitute a protein family of the catalytic subunit of OST along with STT3 from eukaryotes. A structure-aided multiple sequence alignment of the STT3/PglB/AglB protein family revealed three types of OST catalytic centers. This novel classification will provide a useful framework for understanding the enzymatic properties of the OST enzymes from Eukarya, Archaea, and Bacteria.

Nicastrin Is Dispensable for γ-Secretase Protease Activity in the Presence of Specific Presenilin Mutations

γ-Secretase is a multisubunit membrane protein complex consisting of presenilin (PS1), nicastrin (NCT), anterior pharynx-1, and presenilin enhancer 2. To analyze the activity of familial Alzheimer disease mutants and to understand the roles of the subunits, we established a yeast transcriptional activator Gal4p system with artificial γ-secretase substrates containing amyloid precursor protein or Notch fragments. The γ-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4p release from the membrane-bound substrates, i.e. growth of yeast on histidine and adenine, or β-galactosidase assay. We screened and evaluated γ-secretase mutants using this reconstitution system in yeast, which does not possess endogenous γ-secretase activity. When we introduced familial Alzheimer mutants of PS1 in this system, their activities were shown to be loss of function. Although the protease activity of wild type PS1 depends on the other three subunits introduced, we obtained 15 new PS1 mutants, which are active in the absence of NCT. They possessed a S438P mutation at the ninth transmembrane domain (TM9) together with one missense mutation distributed through transmembrane and loop regions. These mutations were not related to familial Alzheimer mutations of PS1 as identified so far. The S438P mutant was partially active but required other mutations for full activation. Results of the β-galactosidase assay suggested that they have wild type protease activities, which were further confirmed by the endoproteolysis of PS1, amyloid β peptides, and Notch intracellular domain production in mammalian cells. These results suggest that NCT is dispensable for the protease activity of γ-secretase.

The Cytochrome c Maturation Components CcmF, CcmH, and CcmI Form a Membrane-integral Multisubunit Heme Ligation Complex

Cytochrome c maturation (Ccm) is a post-translational and post-export protein modification process that involves ten (CcmABCDEFGHI and CcdA or DsbD) components in most Gram-negative bacteria. The absence of any of these components abolishes the ability of cells to form cytochrome c, leading in the case of Rhodobacter capsulatus to the loss of photosynthetic proficiency and respiratory cytochrome oxidase activity. Based on earlier molecular genetic studies, we inferred that R. capsulatus CcmF, CcmH, and CcmI interact with each other to perform heme-apocytochrome c ligation. Here, using functional epitope-tagged derivatives of these components coproduced in appropriate mutant strains, we determined protein-protein interactions between them in detergent-dispersed membranes. Reciprocal affinity purification as well as tandem size exclusion and affinity chromatography analyses provided the first biochemical evidence that CcmF, CcmH, and CcmI associate stably with each other, indicating that these Ccm components form a membrane-integral complex. Under the conditions used, the CcmFHI complex does not contain CcmG, suggesting that the latter thio-reduction component is not always associated with the heme ligation components. The findings are discussed with respect to defining the obligatory components of a minimalistic heme-apocytochrome c ligation complex in R. capsulatus.

Functional Interaction between Paramyxovirus Fusion and Attachment Proteins

Paramyxovirinae envelope glycoproteins constitute a premier model to dissect how specific and dynamic interactions in multisubunit membrane protein complexes can control deep-seated conformational rearrangements. However, individual residues that determine reciprocal specificity of the viral attachment and fusion (F) proteins have not been identified. We have developed an assay based on a pair of canine distemper virus (CDV) F proteins (strains Onderstepoort (ODP) and Lederle) that share approximately 95% identity but differ in their ability to form functional complexes with the measles virus (MV) attachment protein (H). Characterization of CDV F chimeras and mutagenesis reveals four residues in CDV F-ODP (positions 164, 219, 233, and 317) required for productive interaction with MV H. Mutating these residues to the Lederle type disrupts triggering of F-ODP by MV H without affecting functionality when co-expressed with CDV H. Co-immunoprecipitation shows a stronger physical interaction of F-ODP than F-Lederle with MV H. Mutagenesis of MV F highlights the MV residues homologous to CDV F residues 233 and 317 as determinants for physical glycoprotein interaction and fusion activity under homotypic conditions. In assay reversal, the introduction of sections of the CDV H stalk into MV H shows a five-residue fragment (residues 110-114) to mediate specificity for CDV F-Lederle. All of the MV H stalk chimeras are surface-expressed, show hemadsorption activity, and trigger MV F. Combining the five-residue H chimera with the CDV F-ODP quadruple mutant partially restores activity, indicating that the residues identified in either glycoprotein contribute interdependently to the formation of functional complexes. Their localization in structural models of F and H suggests that placement in particular of F residue 233 in close proximity to the 110-114 region of H is structurally conceivable.

The PsbQ protein defines cyanobacterial Photosystem II complexes with highest activity and stability

Light-induced conversion of water to molecular oxygen by Photosystem II (PSII) is one of the most important enzymatic reactions in the biosphere. PSII is a multisubunit membrane protein complex with numerous associated cofactors, but it continually undergoes assembly and disassembly due to frequent light-mediated damage as a result of its normal function. Thus, at any instant, there is heterogeneity in the subunit compositions of PSII complexes within the cell. In particular, cyanobacterial PSII complexes have five associated extrinsic proteins, PsbO, PsbP, PsbQ, PsbU, and PsbV. However, little is known about the interactions of the more recently identified PsbQ protein with other components in cyanobacterial PSII. Here we show that PSII complexes can be isolated from the cyanobacterium Synechocystis sp. PCC 6803 on the basis of the presence of a polyhistidine-tagged PsbQ protein. Purification of PSII complexes using a tagged extrinsic protein has not been previously described, and this work conclusively demonstrates that PsbQ is present in combination with the PsbO, PsbU, and PsbV proteins in cyanobacterial PSII. Moreover, PsbQ-associated PSII complexes have higher activity and stability relative to those isolated using histidine-tagged CP47, an integral membrane protein. Therefore, we conclude that the presence of PsbQ defines the fully assembled and optimally active form of the enzyme.

Two-dimensional Blue Native/SDS Gel Electrophoresis of Multiprotein Complexes from Helicobacter pylori

The study of protein interactions constitutes an important domain to understand the physiology and pathogenesis of microorganisms. The two-dimensional blue native/SDS-PAGE was initially reported to analyze membrane protein complexes. In this study, both cytoplasmic and membrane complexes of a bacterium, the strain J99 of the gastric pathogen Helicobacter pylori, were analyzed by this method. It was possible to identify 34 different proteins grouped in 13 multiprotein complexes, 11 from the cytoplasm and two from the membrane, either previously reported partially or totally in the literature. Besides complexes involved in H. pylori physiology, this method allowed the description of interactions involving known pathogenic factors such as (i) urease with the heat shock protein GroEL or with the putative ketol-acid reductoisomerase IlvC and (ii) the cag pathogenicity island CagA protein with the DNA gyrase GyrA as well as insight on the partners of TsaA, a peroxide reductase/stress-dependent molecular chaperone. The two-dimensional blue native/SDS-PAGE combined with mass spectrometry is a potential tool to study the differences in complexes isolated in various situations and also to study the interactions between bacterial and eucaryotic cell proteins.

STRUCTURE AND FUNCTION OF PHOTOSYSTEMS I AND II

Oxygenic photosynthesis, the principal converter of sunlight into chemical energy on earth, is catalyzed by four multi-subunit membrane-protein complexes: photosystem I (PSI), photosystem II (PSII), the cytochrome b(6)f complex, and F-ATPase. PSI generates the most negative redox potential in nature and largely determines the global amount of enthalpy in living systems. PSII generates an oxidant whose redox potential is high enough to enable it to oxidize H(2)O, a substrate so abundant that it assures a practically unlimited electron source for life on earth. During the last century, the sophisticated techniques of spectroscopy, molecular genetics, and biochemistry were used to reveal the structure and function of the two photosystems. The new structures of PSI and PSII from cyanobacteria, algae, and plants has shed light not only on the architecture and mechanism of action of these intricate membrane complexes, but also on the evolutionary forces that shaped oxygenic photosynthesis.

Structural and functional features of yeast V-ATPase subunit C

V-ATPase is a multi-subunit membrane protein complex, it translocates protons across biological membranes, generating electrical and pH gradients which are used for varieties of cellular processes. V-ATPase is composed of two distinct sub-complexes: a membrane bound V 0 sub-complex, composed of 6 different subunits, which is responsible for proton transport and a soluble cytosolic facing V 1 sub-complex, composed of 8 different subunits which hydrolyse ATP. The two sub-complexes are held together via a flexible stator. One of the main features of eukaryotic V-ATPase is its ability to reversibly dissociate to its sub-complexes in response to changing cellular conditions, which arrest both proton translocation and ATP hydrolysis, suggesting a regulation function. Subunit C (vma5p in yeast) was shown by several biochemical, genetic and recent structural data to function as a flexible stator holding the two sectors of the complex together and regulating the reversible association/dissociation of the complex, partly via association with F-actin filaments. Structural features of subunit C that allow smooth energy conversion and interaction with actin and nucleotides are discussed.

Peripheral Stator of the Yeast V-ATPase: Stoichiometry and Specificity of Interaction between the EG Complex and Subunits C and H

V-ATPases are multisubunit membrane protein complexes that use the energy provided by ATP hydrolysis to generate a proton gradient across various intracellular and plasma membranes. In doing so, they maintain an acidic pH in the lumen of intracellular organelles and acidify extracellular milieu to support specific cellular functions. V-ATPases are structurally similar to the F1F0-ATP synthase, with an intrinsic membrane domain (V0) and an extrinsic peripheral domain (V1) joined by several connecting elements. To gain a clear functional understanding of the catalytic mechanism, and of the stability requirements for regulatory processes in the enzyme, a clear topology of the enzyme has to be established. In particular, the composition and arrangement of the peripheral stator subunits must be firmly settled, as these play specific roles in catalysis and regulation. We have designed a strategy allowing us to coexpress different combinations of these subunits to delineate specific interactions. In this study, we report the interaction between the peripheral stator EG complex and subunits C and H of the V-ATPase from the yeast Saccharomyces cerevisae. A combination of analytical gel filtration, native gel electrophoresis, and ultracentrifugation analysis allowed us to ascertain the homogeneity and molar mass of the purified EGC complex as well as of the EG complex, supporting the formation of 1:1(:1) stoichiometric complexes. The EGC complex can be formed in vitro by combining equimolar amounts of subunit C and the EG subcomplex and results most likely from the initial interaction between subunits E and C.

The structure of photosystem I and evolution of photosynthesis

Oxygenic photosynthesis is the principal producer of both oxygen and organic matter on earth. The primary step in this process--the conversion of sunlight into chemical energy--is driven by four multi-subunit membrane protein complexes named photosystem I, photosystem II, cytochrome b(6)f complex and F-ATPase. Photosystem I generates the most negative redox potential in nature and thus largely determines the global amount of enthalpy in living systems. The recent structural determination of PSI complexes from cyanobacteria and plants sheds light on the evolutionary forces that shaped oxygenic photosynthesis. The fortuitous formation of our solar system in a space plentiful of elements, our distance from the sun and the long time of uninterrupted evolution enabled the perfection of photosynthesis and the evolution of advanced organisms. The available structural information complements the knowledge gained from genomic and proteomic data to illustrate a more precise scenario for the evolution of life systems on earth.

Light-Harvesting Features Revealed by the Structure of Plant Photosystem I

Oxygenic photosynthesis is driven by two multi-subunit membrane protein complexes, Photosystem I and Photosystem II. In plants and green algae, both complexes are composed of two moieties: a reaction center (RC), where light-induced charge translocation occurs, and a peripheral antenna that absorbs light and funnels its energy to the reaction center. The peripheral antenna of PS I (LHC I) is composed of four gene products (Lhca 1-4) that are unique among the chlorophyll a/b binding proteins in their pronounced long-wavelength absorbance and in their assembly into dimers. The recently determined structure of plant Photosystem I provides the first relatively high-resolution structural model of a super-complex containing a reaction center and its peripheral antenna. We describe some of the structural features responsible for the unique properties of LHC I and discuss the advantages of the particular LHC I dimerization mode over monomeric or trimeric forms. In addition, we delineate some of the interactions between the peripheral antenna and the reaction center and discuss how they serve the purpose of dynamically altering the composition of LHC I in response to environmental pressure. Combining structural insight with spectroscopic data, we propose how altering LHC I composition may protect PS I from excessive light.

Crystal structure of plant photosystem I.

Oxygenic photosynthesis is the principal producer of both oxygen and organic matter on Earth. The conversion of sunlight into chemical energy is driven by two multisubunit membrane protein complexes named photosystem I and II. We determined the crystal structure of the complete photosystem I (PSI) from a higher plant (Pisum sativum var. alaska) to 4.4 A resolution. Its intricate structure shows 12 core subunits, 4 different light-harvesting membrane proteins (LHCI) assembled in a half-moon shape on one side of the core, 45 transmembrane helices, 167 chlorophylls, 3 Fe-S clusters and 2 phylloquinones. About 20 chlorophylls are positioned in strategic locations in the cleft between LHCI and the core. This structure provides a framework for exploration not only of energy and electron transfer but also of the evolutionary forces that shaped the photosynthetic apparatus of terrestrial plants after the divergence of chloroplasts from marine cyanobacteria one billion years ago.

Photosystem II: the engine of life.

Photosystem II (PS II) is a multisubunit membrane protein complex, which uses light energy to oxidize water and reduce plastoquinone. High-resolution electron cryomicroscopy and X-ray crystallography are revealing the structure of this important molecular machine. Both approaches have contributed to our understanding of the organization of the transmembrane helices of higher plant and cyanobacterial PS II and both indicate that PS II normally functions as a dimer. However the high-resolution electron density maps derived from X-ray crystallography currently at 3.7/3.8 A, have allowed assignments to be made to the redox active cofactors involved in the light-driven water-plastoquinone oxidoreductase activity and to the chlorophyll molecules that absorb and transfer energy to the reaction centre. In particular the X-ray work has identified density that can accommodate the four manganese atoms which catalyse the water-oxidation process. The Mn cluster is located at the lumenal surface of the DI protein and approximately 7 A from the redox active tyrosine residue (YZ) which acts an electron/proton transfer link to the primary oxidant P680.+. The lower resolution electron microscopy studies, however, are providing structural models of larger PS II supercomplexes that are ideal frameworks in which to incorporate the X-ray derived structures.