scholarly journals Mobility of the SecA 2-helix-finger is not essential for polypeptide translocation via the SecYEG complex

2012 ◽  
Vol 199 (6) ◽  
pp. 919-929 ◽  
Author(s):  
Sarah Whitehouse ◽  
Vicki A.M. Gold ◽  
Alice Robson ◽  
William J. Allen ◽  
Richard B. Sessions ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Translocation ◽  
Energy Transduction ◽  
Power Stroke ◽  
Active Complex ◽  
Essential Protein ◽  
The Core ◽  
Substrate Protein ◽  
Ideal Position ◽  
Protein Channel

The bacterial ATPase SecA and protein channel complex SecYEG form the core of an essential protein translocation machinery. The nature of the conformational changes induced by each stage of the hydrolytic cycle of ATP and how they are coupled to protein translocation are not well understood. The structure of the SecA–SecYEG complex revealed a 2-helix-finger (2HF) of SecA in an ideal position to contact the substrate protein and push it through the membrane. Surprisingly, immobilization of this finger at the edge of the protein channel had no effect on translocation, whereas its imposition inside the channel blocked transport. This analysis resolves the stoichiometry of the active complex, demonstrating that after the initiation process translocation requires only one copy each of SecA and SecYEG. The results also have important implications on the mechanism of energy transduction and the power stroke driving transport. Evidently, the 2HF is not a highly mobile transducing element of polypeptide translocation.

scholarly journals Reovirus core proteins λ1 and σ2 promote stability of disassembly intermediates and influence early replication events

2020 ◽  
Author(s):  
Stephanie Gummersheimer ◽  
Pranav Danthi
Keyword(s):  
Conformational Changes ◽  
Genetic Material ◽  
Point Mutations ◽  
Host Cells ◽  
Capsid Proteins ◽  
Endosomal Membrane ◽  
The Core ◽  
Core Proteins ◽  
Early Replication ◽  
Outer Capsid

ABSTRACTThe capsids of mammalian reovirus contain two concentric protein shells, the core and the outer capsid. The outer capsid is comprised of µ1-σ3 heterohexamers which surround the core. The core is comprised of λ1 decamers held in place by σ2. After entry into the endosome, σ3 is proteolytically degraded and µ1 is cleaved and exposed to form ISVPs. ISVPs undergo further conformational changes to form ISVP*s, resulting in the release of µ1 peptides which facilitate the penetration of the endosomal membrane to release transcriptionally active core particles into the cytoplasm. Previous work has identified regions or specific residues within reovirus outer capsid that impact the efficiency of cell entry. We examined the functions of the core proteins λ1 and σ2. We generated a reovirus T3D reassortant that carries strain T1L derived σ2 and λ1 proteins (T3D/T1L L3S2). This virus displays a lower ISVP stability and therefore converts to ISVP*s more readily. To identify the basis for lability of T3D/T1L L3S2, we screened for hyper-stable mutants of T3D/T1L L3S2 and identified three point mutations in µ1 that stabilize ISVPs. Two of these mutations are located in the C-terminal ϕ region of µ1, which has not previously been implicated in controlling ISVP stability. Independent from compromised ISVP stability, we also found that T3D/T1L L3S2 launches replication more efficiently and produces higher yields in infected cells. In addition to identifying a new role for the core proteins in disassembly events, these data highlight that core proteins may influence multiple stages of infection.IMPORTANCEProtein shells of viruses (capsids) have evolved to undergo specific changes to ensure the timely delivery of genetic material to host cells. The 2-layer capsid of reovirus provides a model system to study the interactions between capsid proteins and the changes they undergo during entry. We tested a virus in which the core proteins were derived from a different strain than the outer capsid. We found that this mismatched virus was less stable and completed conformational changes required for entry prematurely. Capsid stability was restored by introduction of specific changes to the outer capsid, indicating that an optimal fit between inner and outer shells maintains capsid function. Separate from this property, mismatch between these protein layers also impacted the capacity of virus to initiate infection and produce progeny. This study reveals new insights into the roles of capsid proteins and their multiple functions during viral replication.

scholarly journals An Intermolecular π-Stacking Interaction Drives Conformational Changes Necessary to β-Barrel Formation in a Pore-Forming Toxin

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Joshua R. Burns ◽  
Craig J. Morton ◽  
Michael W. Parker ◽  
Rodney K. Tweten
Keyword(s):  
Conformational Changes ◽  
Membrane Attack Complex ◽  
Immune Defense ◽  
Stacking Interaction ◽  
Helical Bundles ◽  
The Core ◽  
Π Stacking ◽  
Π Stacking Interaction ◽  
Membrane Spanning ◽  
Β Sheet

ABSTRACT The crystal structures of the soluble monomers of the pore-forming cholesterol-dependent cytolysins (CDCs) contain two α-helical bundles that flank a twisted core β-sheet. This protein fold is the hallmark of the CDCs, as well as of the membrane attack complex/perforin immune defense proteins and the stonefish toxins. To form the β-barrel pore, a core β-sheet is flattened to align the membrane-spanning β-hairpins. Concomitantly with this conformational change, the two α-helical bundles that flank the core β-sheet break their restraining contacts and refold into two membrane-spanning β-hairpins of the β-barrel pore. The studies herein show that in the monomer structure of the archetype CDC perfringolysin O (PFO), a conserved Met-Met-Phe triad simultaneously contributes to maintaining the twist in this core β-sheet, as well as restricting the α-helical–to–β-strand transition necessary to form one of two membrane-spanning β-hairpins. A previously identified intermolecular π-stacking interaction is now shown to disrupt the interactions mediated by this conserved triad. This is required to establish the subsequent intermolecular electrostatic interaction, which has previously been shown to drive the final conformational changes necessary to form the β-barrel pore. Hence, these studies show that the intermolecular π-stacking and electrostatic interactions work in tandem to flatten the core β-sheet and initiate the α-helical–to–β-strand transitions to form the β-barrel pore. IMPORTANCE A unique feature of the CDC/MACPF/SNTX (cholesterol-dependent cytolysin/membrane attack complex perforin/stonefish toxin) superfamily of pore-forming toxins is that the β-strands that comprise the β-barrel pore are derived from a pair of α-helical bundles. These studies reveal the molecular basis by which the formation of intermolecular interactions within the prepore complex drive the disruption of intramolecular interactions within each monomer of the prepore to trigger the α-helical–to–β-strand transition and formation of the β-barrel pore.

scholarly journals SecA Dimer Cross-Linked at Its Subunit Interface Is Functional for Protein Translocation

2006 ◽  
Vol 188 (1) ◽  
pp. 335-338 ◽  
Author(s):  
Lucia B. Jilaveanu ◽  
Donald Oliver
Keyword(s):  
Conformational Changes ◽  
Protein Transport ◽  
Protein Translocation ◽  
Directed Mutagenesis ◽  
Subunit Interface ◽  
Cargo Proteins ◽  
Considerable Controversy ◽  
The Subject ◽  
Vitro Protein

ABSTRACT SecA facilitates protein transport across the eubacterial plasma membrane by its association with cargo proteins and the SecYEG translocon, followed by ATP-driven conformational changes that promote protein translocation in a stepwise manner. Whether SecA functions as a monomer or a dimer during this process has been the subject of considerable controversy. Here we utilize cysteine-directed mutagenesis along with the crystal structure of the SecA dimer to create a cross-linked dimer at its subunit interface, which was normally active for in vitro protein translocation.

scholarly journals Conformational Changes ofEscherichia coliRNA Polymerase ς70Factor Induced by Binding to the Core Enzyme

1998 ◽  
Vol 273 (49) ◽  
pp. 32995-33001 ◽  
Author(s):  
Sandhya Callaci ◽  
Ewa Heyduk ◽  
Tomasz Heyduk
Keyword(s):  
Conformational Changes ◽  
The Core ◽  
Core Enzyme

scholarly journals Mechanics of torque generation in the bacterial flagellar motor

2015 ◽  
Vol 112 (32) ◽  
pp. E4381-E4389 ◽  
Author(s):  
Kranthi K. Mandadapu ◽  
Jasmine A. Nirody ◽  
Richard M. Berry ◽  
George Oster
Keyword(s):  
Conformational Changes ◽  
Specific Model ◽  
Power Stroke ◽  
Flagellar Motor ◽  
Mechanical Explanation ◽  
Proposed Model ◽  
Torque Generation ◽  
Bacterial Flagellar Motor ◽  
Α Helix ◽  
Fundamental Forces

The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual “power stroke.” Specifically, we propose that ion-induced conformational changes about a proline “hinge” residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque–speed and speed–ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator.

Protein kinases and their therapeutic exploitation

2007 ◽  
Vol 35 (1) ◽  
pp. 7-11 ◽  
Author(s):  
L. Johnson
Keyword(s):  
Protein Kinases ◽  
Conformational Changes ◽  
Glycogen Phosphorylase ◽  
Molecular Level ◽  
Enzyme Activation ◽  
Phosphorylase Kinase ◽  
Binding Properties ◽  
Cyclin Dependent Kinases ◽  
Substrate Protein ◽  
Potential Therapeutic Application

This review focuses on the recognition properties of protein kinases at the molecular level. Phosphorylation of the substrate protein by a protein kinase can result in enzyme activation or inhibition, conformational changes that change recognition properties, or the creation of a surface with distinct binding properties. Protein kinases have become important targets for the development of inhibitors with potential therapeutic application. Various examples are considered in this review, and I discuss our own work on glycogen phosphorylase and phosphorylase kinase, and the structures of proteins involved with the cell cycle, including cyclins and cyclin-dependent kinases.

scholarly journals Forces on nascent polypeptides during membrane insertion and translocation via the Sec translocon

2018 ◽  
Author(s):  
Michiel J.M. Niesen ◽  
Annika Müller-Lucks ◽  
Rickard Hedman ◽  
Gunnar von Heijne ◽  
Thomas F. Miller
Keyword(s):  
Conformational Changes ◽  
Protein Translocation ◽  
Translation Arrest ◽  
Protein Biogenesis ◽  
Pulling Forces ◽  
Transient Interactions ◽  
Pulling Force ◽  
Electrostatic Coupling ◽  
Polypeptide Chains ◽  
Ribosomal Translation

ABSTRACTDuring ribosomal translation, nascent polypeptide chains (NCs) undergo a variety of physical processes that determine their fate in the cell. Translation arrest peptide (AP) experiments are used to measure the external pulling forces that are exerted on the NC at different lengths during translation. To elucidate the molecular origins of these forces, a recently developed coarsegrained molecular dynamics (CGMD) is used to directly simulate the observed pulling-force profiles, thereby disentangling contributions from NC-translocon and NC-ribosome interactions, membrane partitioning, and electrostatic coupling to the membrane potential. This combination of experiment and theory reveals mechanistic features of Sec-facilitated membrane integration and protein translocation, including the interplay between transient interactions and conformational changes that occur during ribosomal translation to govern protein biogenesis.

scholarly journals High-resolution view of the type III secretion export apparatus in situ reveals membrane remodeling and a secretion pathway

2019 ◽  
Author(s):  
Carmen Butan ◽  
Maria Lara-Tejero ◽  
Wenwei Li ◽  
Jun Liu ◽  
Jorge E. Galán
Keyword(s):  
High Resolution ◽  
Protein Secretion ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Protein Translocation ◽  
Membrane Remodeling ◽  
Secretion Systems ◽  
Type Iii ◽  
The Core

AbstractType III protein secretion systems are essential virulence factors for many important pathogenic bacteria. The entire protein secretion machine is composed of several substructures that organize into a holostructure or injectisome. The core component of the injectisome is the needle complex, which houses the export apparatus that serves as a gate for the passage of the secreted proteins through the bacterial inner membrane. Here we describe a high-resolution structure of the export apparatus of the Salmonella type III secretion system in association with the needle complex and the underlying bacterial membrane, both in isolation and in situ. We show the precise location of the core export apparatus components within the injectisome and bacterial envelope and demonstrate that their deployment results in major membrane remodeling and thinning, which may be central for the protein translocation process. We also show that InvA, a critical export apparatus component, forms a multi-ring cytoplasmic conduit that provides a pathway for the type III secretion substrates to reach the entrance of the export gate. Combined with structure-guided mutagenesis, our studies provide major insight into potential mechanisms of protein translocation and injectisome assembly.

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