Helical Gate Research Articles

Motif V regulates energy transduction between the flavivirus NS3 ATPase and RNA-binding cleft

The unwinding of dsRNA intermediates is critical for the replication of flavivirus RNA genomes. This activity is provided by the C-terminal helicase domain of viral nonstructural protein 3 (NS3). As a member of the superfamily 2 (SF2) helicases, NS3 requires the binding and hydrolysis of ATP/NTP to translocate along and unwind double-stranded nucleic acids. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well-understood. Previous molecular dynamics simulations conducted by our group have identified Motif V as a potential "communication hub" for this energy transduction pathway. To investigate the role of Motif V in this process, here we combined molecular dynamics, biochemistry, and virology approaches. We tested Motif V mutations in both the replicon and recombinant protein systems to investigate viral genome replication, RNA-binding affinity, ATP hydrolysis activity, and helicase-mediated unwinding activity. We found that the T407A and S411A substitutions in NS3 reduce viral replication and increase the helicase-unwinding turnover rates by 1.7- and 3.5-fold, respectively, suggesting that flaviviruses may use suboptimal NS3 helicase activity for optimal genome replication. Additionally, we used simulations of each mutant to probe structural changes within NS3 caused by each mutation. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. These results help define the linkage between ATP hydrolysis and helicase activities within NS3 and provide insight into the biophysical mechanisms for ATPase-driven NS3 helicase function.

Role of Lipids in the Reaction Coordinate of GlpG Rhomboid Protease

Rhomboid proteases function in hydrated lipid membrane environments that can impact significantly the catalytic activity of the enzyme. How lipids participate in the reaction coordinate of rhomboid proteases is, however, unclear. We address this question by performing all-atom molecular dynamics simulations of the rhomboid protease from Escherichia coli, GlpG. We study GlpG starting from three different protein crystal structures, and explore interactions with lipids by considering five one-component lipid membranes with different lipid headgroups or alkyl chains, and in mixed membranes of phosphatidylethanolamine and phosphatidylgylcerol lipids. Lipid alkyl chains can visit inter-helical regions of the protein close to the gate helix, which can indicate regions of potential interest for substrate binding. Variability in how the protein can interact with lipids closest to the helical gate region suggests that membrane environments may impact proteolysis by participating in the substrate-binding step of the reaction coordinate.Research was supported in part by funding from the Excellence Initiative of the German Federal and State Governments via the Freie Universitat Berlin, and by an allocation of computing time from HLRN, The North-German Supercomputing Center (bec00076).

Structures of the Human HCN1 Hyperpolarization-Activated Channel

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the control of rhythmic activity in cardiac and neuronal pacemaker cells. In HCN, the polarity of voltage dependence is uniquely reversed. Intracellular cyclic adenosine monophosphate (cAMP) levels tune the voltage response, enabling sympathetic nerve stimulation to increase the heart rate. We present cryo-electron microscopy structures of the human HCN channel in the absence and presence of cAMP at 3.5Å resolution. HCN channels contain a K+ channel selectivity filter-forming sequence from which the amino acids create a unique structure that explains Na+ and K+ permeability. The voltage sensor adopts a depolarized conformation, and the pore is closed. An S4 helix of unprecedented length extends into the cytoplasm, contacts the C-linker, and twists the inner helical gate shut. cAMP binding rotates cytoplasmic domains to favor opening of the inner helical gate. These structures advance understanding of ion selectivity, reversed polarity gating, and cAMP regulation in HCN channels.

Of GIRK channels, vitamin E transfer, and a vertebrate fluorescent protein

Of GIRK channels, vitamin E transfer, and a vertebrate fluorescent protein

Long Distance Lipid: Protein Coupling in the Protein Translocon

The SecY/Sec61 protein translocon is the central component of the sophisticated translocase machinery essential for the biosynthesis of membrane and secreted proteins in all branches of life. To incorporate newly synthesized transmembrane (TM) proteins into the lipid membrane, SecY opens a helical gate formed by TM helices 2 and 7 towards the bilayer. Opening of a lateral helical gate to release TM proteins requires close communication between the translocon and the lipid membrane.Here we report prolonged molecular dynamics computations on wild-type and mutant translocons with perturbed lipid interactions. We find a remarkable long-distance coupling between the translocon and the surrounding lipid membrane. Mutating amino acids that in the wild type interact with lipids affects the shape of the lipid membrane surrounding the translocon. Importantly, the status of the lipid:translocon interactions is rapidly communicated to the plug - a structural element whose displacement is thought to be part of the translocon opening. The rapid propagation of structural perturbations throughout the translocon is likely facilitated by the extensive hydrogen-bonding network of the translocon. The tight coupling between the translocon and the lipid membrane demonstrated here could explain the experimental observations that the lipid membrane composition affects the proper assembly and functioning of the translocase machinery.Research supported in part by the National Institute of General Medical Sciences (GM-74637 and GM-86685 to S.H.W), the NSF (CHE-0750175 to D.J.T), and an allocation of computer time from the NSF through the TeraGrid resources at TACC (Ranger). A.-N.B is supported in part by Marie Curie International Reintegration Award FP7-PEOPLE-2010-RG 276920.

A blueprint for functional engineering: Single point mutations reconstitute phosphatidylinositol presentation in a pseudo-Sec14 protein

Phosphoinositides, phosphorylated species of phosphatidylinositol (PtdIns), are critical regulatory lipids in all eukaryotic cells. The molecular mechanisms that lead to the phosphorylation of an individual PtdIns- or phosphoinositide molecule remain largely unkown even though lipid kinases and phosphatases involved in these processes have been studied in detail. The observation by us and others that liposomal PtdIns (and phosphoinositide) molecules are poor in vitro substrates for kinases and phosphatases raises the question of how these enzymes execute their function in living cells. Recent work indicates that Sec14, the founding member of a large superfamily of eukaryotic proteins, is crucial for the process of PtdIns phosphorylation. The collective data suggest that Sec14 mediates a heterotypic phospholipid exchange reaction of PtdIns with phosphatidylcholine (PtdCho) during which PtdIns becomes vulnerable for kinase attack and thereby promotes the generation of phosphoinositides.1,2 In a recent paper we address the molecular mechanism of this phospholipid (PL) exchange reaction in a pseudo-Sec14 protein (Sfh1) that we rendered functional by a directed evolution approach. We find that enhanced PL-cycling into and out of the hydrophobic pocket of these activated Sfh1 mutants depends on the reconfiguration of interactions between a C-terminal string motif and the floor of the hydrophobic pocket that results in increased oscillations in a helical gate that controls pocket access. Here we further discuss our findings and propose molecular dynamics simulations as a tool to approach energetically unfavorable transition states and to identify novel protein-ligand interactions invisible to X-ray crystallography.

Lipid-Mediated Helix Gating in the GlpG Rhomboid Protease from Escherichia Coli

Intramembrane proteases cleave transmembrane substrates to liberate molecules that participate in essential cellular processes, such as cell signaling and gene regulation. Understanding how intramembrane proteases work requires knowledge of how substrates are docked and cleaved within the membrane plane. We find that lipid interactions and the presence of the substrate affect significantly the structure and dynamics of the helical gate and of the cap loop that control access to the active site of the GlpG rhomboid protease from E. coli.In the absence of the substrate, the 1-palmytoyl-2-oleoyl-sn-glycero-3-phosphatidyethanolamine (POPE) lipid molecule bound to the protease active site remains closely associated with the protease on the ∼90ns timescale of the molecular dynamics (MD) simulation. Presence of the Spitz transmembrane substrate perturbs the interactions between the protease and the active-site lipid molecule. The substrate and whether or not a lipid headgroup is bound to the active site affect the orientation of the lateral gate helix 5 relative to the rest of the protein, the structure and dynamics of the cap loop, and the interaction between the catalytic serine and water. This suggests that lipid molecules may be involved in controlling the access to the active site of intramembrane proteases.This research was supported in part by the National Institute of General Medical Sciences (GM-74637 and GM-86685 to S.H.W) and an allocation of computer time from the National Science Foundation through the TeraGrid resources at TACC (Ranger).

Opening the SecYEG Protein Translocon

The Sec61/SecYEG translocon is the central component of the major biosynthetic pathway for membrane and secreted proteins. To incorporate membrane proteins into the endoplasmic reticulum membrane of eukaryotes (Sec61), or into the plasma membrane of prokaryotes (SecYEG), the translocon relies on the opening of a lateral helical gate formed by transmembrane (TM) helices TM2 and TM7. The crystallographic structure of the closed state of the translocon (B. van der Berg et al, Nature 427, 36-44, 2003) solved in the absence of bound signal peptide provides valuable information on possible scenarios of membrane protein insertion. Nevertheless, the sequence of structural rearrangements that leads to the opening of the translocon and the geometry of the translocon in its open state remain unknown. To derive information on the structure and dynamics of possible open conformations of the SecYEG translocon, we performed prolonged molecular dynamics simulations of the SecYEG translocon in which we mutated residues determined to participate in stabilizing interactions of the closed state. We have also performed a simulation of the SecYEG translocon with a bound signal peptide. The computations provide insights into interactions essential for keeping the lateral gate closed and how perturbations of these interactions cause rearrangements of the gate.This work was supported by research grants from the National Institute for General Medical Sciences, the NIH National Center for Research Resources, and the National Science Foundation.

A Relay Mechanism in Rhomboid Intramembrane Protease

Intramembrane proteases are a family of highly conserved membrane proteins that cleave other transmembrane (TM) helical segments within the plane of the lipid membrane. GlpG rhomboid protease, the best characterized of these intramembrane proteases, has six helical segments and a unique loop lying in the plane of the membrane. TM5 acts as the lateral gate that opens to allow docking of the incoming substrate; the role of the L1 loop, which extends away from the active site, is uncertain. Site-directed mutagenesis experiments have identified a triple serine L1 mutant (Y138S/F139S/L143S) with a significantly reduced catalytic activity, and a triple valine mutant of the TM5 gate (L229V/F232V/W236V) with enhanced activity relative to the wild-type protease (Baker et al, Proc. Natl. Acad. Sci. USA 104, 8257-8262, 2007). To dissect the roles of TM5 and L1, we performed all-atom molecular dynamics simulations of the L1 and TM5 mutants in hydrated bilayers of 1-palmytoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE). The results reveal a relay mechanism that transmits structural and dynamical perturbations between the remote TM5/L1 structural elements of the protease. Perturbation of L1 is transmitted to the active site and TM5 via intra-protein hydrogen bonds to which conserved amino acid residues contribute. Likewise, perturbation of TM5 leads to changes in protein dynamics and local structural rearrangements of the remote L1 loop. In the (inactive) L1 triple serine mutant, but not in the (highly active) TM5 triple valine mutant, several intra-protein interactions become locked in a new geometry. The communication between L1 and the TM5 helical gate TM5 suggests a regulatory role for loop L1.This work was supported by research grants from the National Institute for General Medical Sciences and the NIH National Center for Research Resources.

Conformational dynamics of the major yeast phosphatidylinositol transfer protein sec14p: insight into the mechanisms of phospholipid exchange and diseases of sec14p-like protein deficiencies.

Molecular dynamics simulations coupled with functional analyses of the major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p identify structural elements involved in regulating the ability of Sec14p to execute phospholipid exchange. The molecular dynamics simulations suggest large rigid body motions within the Sec14p molecule accompany closing and opening of an A(10)/T(4)/A(11) helical gate, and that "state-of-closure" of this helical gate determines access to the Sec14p phospholipid binding cavity. The data also project that conformational dynamics of the helical gate are controlled by a hinge unit (residues F(212), Y(213), K(239), I(240), and I(242)) that links to the N- and C-terminal ends of the helical gate, and by a novel gating module (composed of the B(1)LB(2) and A(12)LT(5) substructures) through which conformational information is transduced to the hinge. The (114)TDKDGR(119) motif of B(1)LB(2) plays an important role in that transduction process. These simulations offer new mechanistic possibilities for an important half-reaction of the Sec14p phospholipid exchange cycle that occurs on membrane surfaces after Sec14p has ejected bound ligand, and is reloading with another phospholipid molecule. These conformational transitions further suggest structural rationales for known disease missense mutations that functionally compromise mammalian members of the Sec14-protein superfamily.