A role for the two-helix finger of the SecA ATPase in protein translocation

An important step in the biosynthesis of many proteins is their partial or complete translocation across the plasma membrane in prokaryotes or the endoplasmic reticulum membrane in eukaryotes. In bacteria, secretory proteins are generally translocated after completion of their synthesis by the interaction of the cytoplasmic ATPase SecA and a protein-conducting channel formed by the SecY complex. How SecA moves substrates through the SecY channel is unclear. However, a recent structure of a SecA-SecY complex raises the possibility that the polypeptide chain is moved by a two-helix finger domain of SecA that is inserted into the cytoplasmic opening of the SecY channel. Here we have used disulphide-bridge crosslinking to show that the loop at the tip of the two-helix finger of Escherichia coli SecA interacts with a polypeptide chain right at the entrance into the SecY pore. Mutagenesis demonstrates that a tyrosine in the loop is particularly important for translocation, but can be replaced by some other bulky, hydrophobic residues. We propose that the two-helix finger of SecA moves a polypeptide chain into the SecY channel with the tyrosine providing the major contact with the substrate, a mechanism analogous to that suggested for hexameric, protein-translocating ATPases.     

Conformational transition of Sec machinery inferred from bacterial SecYE structures

Over 30% of proteins are secreted across or integrated into membranes. Their newly synthesized forms contain either cleavable signal sequences or non-cleavable membrane anchor sequences, which direct them to the evolutionarily conserved Sec translocon (SecYEG in prokaryotes and Sec61, comprising alpha-, gamma- and beta-subunits, in eukaryotes). The translocon then functions as a protein-conducting channel. These processes of protein localization occur either at or after translation. In bacteria, the SecA ATPase drives post-translational translocation. The only high-resolution structure of a translocon available so far is that for SecYEbeta from the archaeon Methanococcus jannaschii, which lacks SecA. Here we present the 3.2-A-resolution crystal structure of the SecYE translocon from a SecA-containing organism, Thermus thermophilus. The structure, solved as a complex with an anti-SecY Fab fragment, revealed a 'pre-open' state of SecYE, in which several transmembrane helices are shifted, as compared to the previous SecYEbeta structure, to create a hydrophobic crack open to the cytoplasm. Fab and SecA bind to a common site at the tip of the cytoplasmic domain of SecY. Molecular dynamics and disulphide mapping analyses suggest that the pre-open state might represent a SecYE conformational transition that is inducible by SecA binding. Moreover, we identified a SecA-SecYE interface that comprises SecA residues originally buried inside the protein, indicating that both the channel and the motor components of the Sec machinery undergo cooperative conformational changes on formation of the functional complex.     

Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes

A decisive step in the biosynthesis of many proteins is their partial or complete translocation across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. Most of these proteins are translocated through a protein-conducting channel that is formed by a conserved, heterotrimeric membrane-protein complex, the Sec61 or SecY complex. Depending on channel binding partners, polypeptides are moved by different mechanisms: the polypeptide chain is transferred directly into the channel by the translating ribosome, a ratcheting mechanism is used by the endoplasmic reticulum chaperone BiP, and a pushing mechanism is used by the bacterial ATPase SecA. Structural, genetic and biochemical data show how the channel opens across the membrane, releases hydrophobic segments of membrane proteins laterally into lipid, and maintains the membrane barrier for small molecules.     

X-ray structure of a protein-conducting channel

A conserved heterotrimeric membrane protein complex, the Sec61 or SecY complex, forms a protein-conducting channel, allowing polypeptides to be transferred across or integrated into membranes. We report the crystal structure of the complex from Methanococcus jannaschii at a resolution of 3.2 A. The structure suggests that one copy of the heterotrimer serves as a functional translocation channel. The alpha-subunit has two linked halves, transmembrane segments 1-5 and 6-10, clamped together by the gamma-subunit. A cytoplasmic funnel leading into the channel is plugged by a short helix. Plug displacement can open the channel into an 'hourglass' with a ring of hydrophobic residues at its constriction. This ring may form a seal around the translocating polypeptide, hindering the permeation of other molecules. The structure also suggests mechanisms for signal-sequence recognition and for the lateral exit of transmembrane segments of nascent membrane proteins into lipid, and indicates binding sites for partners that provide the driving force for translocation.     

Structural basis of PIP2 activation of the classical inward rectifier K+ channel Kir2.2

The regulation of ion channel activity by specific lipid molecules is widely recognized as an integral component of electrical signalling in cells. In particular, phosphatidylinositol 4,5-bisphosphate (PIP(2)), a minor yet dynamic phospholipid component of cell membranes, is known to regulate many different ion channels. PIP(2) is the primary agonist for classical inward rectifier (Kir2) channels, through which this lipid can regulate a cell's resting membrane potential. However, the molecular mechanism by which PIP(2) exerts its action is unknown. Here we present the X-ray crystal structure of a Kir2.2 channel in complex with a short-chain (dioctanoyl) derivative of PIP(2). We found that PIP(2) binds at an interface between the transmembrane domain (TMD) and the cytoplasmic domain (CTD). The PIP(2)-binding site consists of a conserved non-specific phospholipid-binding region in the TMD and a specific phosphatidylinositol-binding region in the CTD. On PIP(2) binding, a flexible expansion linker contracts to a compact helical structure, the CTD translates 6 ? and becomes tethered to the TMD and the inner helix gate begins to open. In contrast, the small anionic lipid dioctanoyl glycerol pyrophosphatidic acid (PPA) also binds to the non-specific TMD region, but not to the specific phosphatidylinositol region, and thus fails to engage the CTD or open the channel. Our results show how PIP(2) can control the resting membrane potential through a specific ion-channel-receptor-ligand interaction that brings about a large conformational change, analogous to neurotransmitter activation of ion channels at synapses.     

Three-dimensional structure of the bacterial protein-translocation complex SecYEG

Transport and membrane integration of polypeptides is carried out by specific protein complexes in the membranes of all living cells. The Sec transport path provides an essential and ubiquitous route for protein translocation. In the bacterial cytoplasmic membrane, the channel is formed by oligomers of a heterotrimeric membrane protein complex consisting of subunits SecY, SecE and SecG. In the endoplasmic reticulum membrane, the channel is formed from the related Sec61 complex. Here we report the structure of the Escherichia coli SecYEG assembly at an in-plane resolution of 8 A. The three-dimensional map, calculated from two-dimensional SecYEG crystals, reveals a sandwich of two membranes interacting through the extensive cytoplasmic domains. Each membrane is composed of dimers of SecYEG. The monomeric complex contains 15 transmembrane helices. In the centre of the dimer we observe a 16 x 25 A cavity closed on the periplasmic side by two highly tilted transmembrane helices. This may represent the closed state of the protein-conducting channel.     

Preserving the membrane barrier for small molecules during bacterial protein translocation

Many proteins are translocated through the SecY channel in bacteria and archaea and through the related Sec61 channel in eukaryotes. The channel has an hourglass shape with a narrow constriction approximately halfway across the membrane, formed by a pore ring of amino acids. While the cytoplasmic cavity of the channel is empty, the extracellular cavity is filled with a short helix called the plug, which moves out of the way during protein translocation. The mechanism by which the channel transports large polypeptides and yet prevents the passage of small molecules, such as ions or metabolites, has been controversial. Here, we have addressed this issue in intact Escherichia coli cells by testing the permeation of small molecules through wild-type and mutant SecY channels, which are either in the resting state or contain a defined translocating polypeptide chain. We show that in the resting state, the channel is sealed by both the pore ring and the plug domain. During translocation, the pore ring forms a 'gasket-like' seal around the polypeptide chain, preventing the permeation of small molecules. The structural conservation of the channel in all organisms indicates that this may be a universal mechanism by which the membrane barrier is maintained during protein translocation.     

Crystal structure of the FimD usher bound to its cognate FimC-FimH substrate

Type 1 pili are the archetypal representative of a widespread class of adhesive multisubunit fibres in Gram-negative bacteria. During pilus assembly, subunits dock as chaperone-bound complexes to an usher, which catalyses their polymerization and mediates pilus translocation across the outer membrane. Here we report the crystal structure of the full-length FimD usher bound to the FimC-FimH chaperone-adhesin complex and that of the unbound form of the FimD translocation domain. The FimD-FimC-FimH structure shows FimH inserted inside the FimD 24-stranded β-barrel translocation channel. FimC-FimH is held in place through interactions with the two carboxy-terminal periplasmic domains of FimD, a binding mode confirmed in solution by electron paramagnetic resonance spectroscopy. To accommodate FimH, the usher plug domain is displaced from the barrel lumen to the periplasm, concomitant with a marked conformational change in the β-barrel. The amino-terminal domain of FimD is observed in an ideal position to catalyse incorporation of a newly recruited chaperone-subunit complex. The FimD-FimC-FimH structure provides unique insights into the pilus subunit incorporation cycle, and captures the first view of a protein transporter in the act of secreting its cognate substrate.     

Crystal structure of the β2 adrenergic receptor-Gs protein complex

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the β(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β(2)AR include a 14 ? outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.     

Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain

The conserved formin homology 2 (FH2) domain nucleates actin filaments and remains bound to the barbed end of the growing filament. Here we report the crystal structure of the yeast Bni1p FH2 domain in complex with tetramethylrhodamine-actin. Each of the two structural units in the FH2 dimer binds two actins in an orientation similar to that in an actin filament, suggesting that this structure could function as a filament nucleus. Biochemical properties of heterodimeric FH2 mutants suggest that the wild-type protein equilibrates between two bound states at the barbed end: one permitting monomer binding and the other permitting monomer dissociation. Interconversion between these states allows processive barbed-end polymerization and depolymerization in the presence of bound FH2 domain. Kinetic and/or thermodynamic differences in the conformational and binding equilibria can explain the variable activity of different FH2 domains as well as the effects of the actin-binding protein profilin on FH2 function.     

Structure of human follicle-stimulating hormone in complex with its receptor

Follicle-stimulating hormone (FSH) is central to reproduction in mammals. It acts through a G-protein-coupled receptor on the surface of target cells to stimulate testicular and ovarian functions. We present here the 2.9-A-resolution structure of a partially deglycosylated complex of human FSH bound to the extracellular hormone-binding domain of its receptor (FSHR(HB)). The hormone is bound in a hand-clasp fashion to an elongated, curved receptor. The buried interface of the complex is large (2,600 A2) and has a high charge density. Our analysis suggests that all glycoprotein hormones bind to their receptors in this mode and that binding specificity is mediated by key interaction sites involving both the common alpha- and hormone-specific beta-subunits. On binding, FSH undergoes a concerted conformational change that affects protruding loops implicated in receptor activation. The FSH-FSHR(HB) complexes form dimers in the crystal and at high concentrations in solution. Such dimers may participate in transmembrane signal transduction.     

Following the signal sequence from ribosomal tunnel exit to signal recognition particle

Membrane and secretory proteins can be co-translationally inserted into or translocated across the membrane. This process is dependent on signal sequence recognition on the ribosome by the signal recognition particle (SRP), which results in targeting of the ribosome-nascent-chain complex to the protein-conducting channel at the membrane. Here we present an ensemble of structures at subnanometre resolution, revealing the signal sequence both at the ribosomal tunnel exit and in the bacterial and eukaryotic ribosome-SRP complexes. Molecular details of signal sequence interaction in both prokaryotic and eukaryotic complexes were obtained by fitting high-resolution molecular models. The signal sequence is presented at the ribosomal tunnel exit in an exposed position ready for accommodation in the hydrophobic groove of the rearranged SRP54 M domain. Upon ribosome binding, the SRP54 NG domain also undergoes a conformational rearrangement, priming it for the subsequent docking reaction with the NG domain of the SRP receptor. These findings provide the structural basis for improving our understanding of the early steps of co-translational protein sorting.     

Crystal structure of metarhodopsin II

G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular heterotrimeric G proteins (Gαβγ). The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta?II) is formed, in which the retinal Schiff base is still intact but deprotonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplasmic crevice for uptake of the interacting C terminus of the Gα subunit. Owing to Schiff base hydrolysis, Meta?II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta?II, presuming that the crystal's high concentration of opsin in an active conformation (Ops*) may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0?? and 2.85?? crystal structures, respectively, of Meta?II alone or in complex with an 11-amino-acid C-terminal fragment derived from Gα (GαCT2). GαCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Gα-derived peptide to Ops* (ref. 7). In the Meta?II structures, the electron density from the retinal ligand seamlessly continues into the Lys?296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all-trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta?II. The structures can now serve as models for the large GPCR family.     

Structure of the guide-strand-containing argonaute silencing complex

The slicer activity of the RNA-induced silencing complex is associated with argonaute, the RNase H-like PIWI domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-base DNA guide strand, thereby identifying the nucleic-acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both of its ends, with the solvent-exposed Watson-Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas two critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies indicate that key amino acid residues at the active site and those lining the 5'-phosphate-binding pocket made up of the Mid domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3'-end-binding pocket made up of the PAZ domain show little effect.     

Mechanism of glutamate receptor desensitization

Ligand-gated ion channels transduce chemical signals into electrical impulses by opening a transmembrane pore in response to binding one or more neurotransmitter molecules. After activation, many ligand-gated ion channels enter a desensitized state in which the neurotransmitter remains bound but the ion channel is closed. Although receptor desensitization is crucial to the functioning of many ligand-gated ion channels in vivo, the molecular basis of this important process has until now defied analysis. Using the GluR2 AMPA-sensitive glutamate receptor, we show here that the ligand-binding cores form dimers and that stabilization of the intradimer interface by either mutations or allosteric modulators reduces desensitization. Perturbations that destabilize the interface enhance desensitization. Receptor activation involves conformational changes within each subunit that result in an increase in the separation of portions of the receptor that are linked to the ion channel. Our analysis defines the dimer interface in the resting and activated state, indicates how ligand binding is coupled to gating, and suggests modes of dimer dimer interaction in the assembled tetramer. Desensitization occurs through rearrangement of the dimer interface, which disengages the agonist-induced conformational change in the ligand-binding core from the ion channel gate.     

Amino-acid sequence of a Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence

We have cloned and sequenced complementary DNA encoding a Ca2+-ATPase of rabbit muscle sarcoplasmic reticulum. We propose a model of the protein which has 3 cytoplasmic domains joined to a set of 10 transmembrane helices by a narrow, penta-helical stalk. In this model, ATP bound to one cytoplasmic domain would phosphorylate an aspartate in an adjoining cytoplasmic domain, inducing translocation of Ca2+ from binding sites on the stalk.     

别构酶ATCase与底物分子协同结合过程的构象变化特性

利用可描述协同结合特性的动力学方法,得到了反映单个别构酶ATCase分子的结合分数与底物浓度之间的关系式.表明在确定的底物浓度条件下,单个别构酶ATCase分子处于确定的构象态,随着底物浓度的增加,别构酶ATCase分子将经历一系列中间态达到饱和构象态,S型结合分数曲线实际上显示了单个别构酶ATCase分子与底物分子的协同结合特性.利用所得模型分析了调节物分子对底物协同结合过程的影响,合理解释了结合分数曲线随调节物浓度变化而发生改变的实验结果,与别构酶ATCase分子具有的结构及所产生的生物学功能是自给的.     

A mutation that prevents GTP-dependent activation of the alpha chain of Gs

Membrane-bound G proteins carry information from receptors on the outside of cells to effector proteins inside cells. The alpha subunits of these heterotrimeric proteins bind and hydrolyse GTP and control the specificity of interactions with receptor and effector elements. Signalling by G proteins involves a cycle in which the inactive alpha beta gamma-GDP complex dissociates to produce alpha*-GTP, which is capable of activating the effector enzyme or ion channel; the alpha*-GTP complex hydrolyses bound GTP and reassociates with beta gamma to form the inactive complex. We have characterized a mutation that interrupts this GTP-driven cycle in alpha s, the alpha-chain of Gs, the G protein that stimulates adenylyl cyclase. The mutation converts a glycine to an alanine residue in the presumed GDP-binding domain of alpha s. The location and biochemical consequences of this mutation suggest a common mechanism by which binding of GTP or ATP may induce changes in the conformation of a number of nucleoside triphosphate binding proteins.     

Conformational changes associated with ion permeation in L-type calcium channels

The mechanism by which ions deliver their message to effector proteins involves a change in the protein conformation which is induced by the specific interaction of the ion with its binding site on the protein. In the case of an ion-channel protein, conformational changes induced by permeant ions and the consequences for channel function have received little attention. Here we report that binding of permeant cations to an intra-channel binding site of the dihydropyridine (DHP)-sensitive (L-type) Ca2+ channel leads to a conformational change which destabilizes the protonated state of a group on the external channel surface, and can shift its apparent pK value by more than 2 pH units. The lifetime of the protonated state correlates with the occupancy of an intra-channel binding site by the permeant cation. The demonstration of such conformational changes in a channel protein induced by the permeant ion has important implications for realistic models of the mechanism of ion permeation.     

Crystal structure of the CorA Mg2+ transporter

The magnesium ion, Mg2+, is essential for myriad biochemical processes and remains the only major biological ion whose transport mechanisms remain unknown. The CorA family of magnesium transporters is the primary Mg2+ uptake system of most prokaryotes and a functional homologue of the eukaryotic mitochondrial magnesium transporter. Here we determine crystal structures of the full-length Thermotoga maritima CorA in an apparent closed state and its isolated cytoplasmic domain at 3.9 A and 1.85 A resolution, respectively. The transporter is a funnel-shaped homopentamer with two transmembrane helices per monomer. The channel is formed by an inner group of five helices and putatively gated by bulky hydrophobic residues. The large cytoplasmic domain forms a funnel whose wide mouth points into the cell and whose walls are formed by five long helices that are extensions of the transmembrane helices. The cytoplasmic neck of the pore is surrounded, on the outside of the funnel, by a ring of highly conserved positively charged residues. Two negatively charged helices in the cytoplasmic domain extend back towards the membrane on the outside of the funnel and abut the ring of positive charge. An apparent Mg2+ ion was bound between monomers at a conserved site in the cytoplasmic domain, suggesting a mechanism to link gating of the pore to the intracellular concentration of Mg2+.