Nucleotide-dependent Conformational Changes Research Articles

Structural insights into the activation mechanism of antimicrobial GBP1

The dynamin-related human guanylate-binding protein 1 (GBP1) mediates host defenses against microbial pathogens. Upon GTP binding and hydrolysis, auto-inhibited GBP1 monomers dimerize and assemble into soluble and membrane-bound oligomers, which are crucial for innate immune responses. How higher-order GBP1 oligomers are built from dimers, and how assembly is coordinated with nucleotide-dependent conformational changes, has remained elusive. Here, we present cryo-electron microscopy-based structural data of soluble and membrane-bound GBP1 oligomers, which show that GBP1 assembles in an outstretched dimeric conformation. We identify a surface-exposed helix in the large GTPase domain that contributes to the oligomerization interface, and we probe its nucleotide- and dimerization-dependent movements that facilitate the formation of an antimicrobial protein coat on a gram-negative bacterial pathogen. Our results reveal a sophisticated activation mechanism for GBP1, in which nucleotide-dependent structural changes coordinate dimerization, oligomerization, and membrane binding to allow encapsulation of pathogens within an antimicrobial protein coat.

Characterizing a novel CMK-EngA fusion protein from Bifidobacterium: Implications for inter-domain regulation

EngA is an essential and unique bacterial GTPase involved in ribosome biogenesis. The essentiality and species-specific variations among EngA homologues make the protein a potential target for future drug development. In this aspect, it is important to understand the variations of EngA among probiotic organisms and non-probiotic bacteria to understand species specificity. The search for variations among EngA homologues revealed a unique variant, exclusively found in Bifidobacterium and a few Actinobacteria species. Bifidobacterium possesses a multifunctional fusion protein, wherein EngA is fused with an N-terminal CMK (Cytidylate Monophosphate Kinase) domain. The resulting protein is therefore a large (70kDa size) with 3 consecutive P-loops and a 50 amino acid long linker connecting the EngA and CMK domains. EngA is known to regulate ribosome biogenesis via nucleotide-dependent conformational changes. The additional domain may introduce further intricate regulation in ribosome biogenesis or participate in newer biological processes. This study is the first attempt to characterise this novel class of bacterial EngA found in the Genus of Bifidobacteria.

Coarse grained modelling highlights the binding differences in the two different allosteric sites of the Human Kinesin EG5 and its implications in inhibitor design

Kinesins involved in mitotic cell division have gained prominence as promising chemotherapy targets. One such kinesin, EG5, a motor protein responsible for cell division, is a validated chemotherapy target with several compounds at various stages of clinical trials. EG5 has an active site and two different allosteric sites that are known to have ligand specificity. Upon ligand binding, EG5's motor domain will no longer undergo nucleotide-dependent conformational changes required to complete the catalytic cycle. However, there is a lack of in-depth knowledge on the mechanism of inhibitor binding to the two different allosteric sites. To understand the EG5's inhibition mechanism and interactions at allosteric sites and other functionally important regions, we generated two coarse-grained models, Gaussian Network Model (GNM) and Anisotropic Network Model (ANM), to identify the dynamics and its correlation to EG5's function. The first three slowest modes of GNM showed marked differences between the various models of EG5. In the first mode, when the inhibitor is bound at allosteric site 1, there is a presence of a hinge region around residue 166, which is not found when the inhibitor is bound at allosteric site 2 or allosteric sites 1 and 2. The third slowest mode showed a distinctive positively correlated region when the inhibitor is bound at allosteric site 2. These differences indicated that the mechanism of binding at allosteric site 1 and allosteric site 2 are unique. Further, it was observed that the simultaneous ligand binding at allosteric sites 1 and 2 shares structural dynamics and interactions that were found while ligand binds at allosteric sites 1 and 2 independently, leading to a new mechanism. Taken together, our observations suggest that there are different mechanisms at play in each inhibitor bound system considered.

Molecular Recognition at Septin Interfaces: The Switches Hold the Key

The assembly of a septin filament requires that homologous monomers must distinguish between one another in establishing appropriate interfaces with their neighbors. To understand this phenomenon at the molecular level, we present the first four crystal structures of heterodimeric septin complexes. We describe in detail the two distinct types of G-interface present within the octameric particles, which must polymerize to form filaments. These are formed between SEPT2 and SEPT6 and between SEPT7 and SEPT3, and their description permits an understanding of the structural basis for the selectivity necessary for correct filament assembly. By replacing SEPT6 by SEPT8 or SEPT11, it is possible to rationalize Kinoshita's postulate, which predicts the exchangeability of septins from within a subgroup. Switches I and II, which in classical small GTPases provide a mechanism for nucleotide-dependent conformational change, have been repurposed in septins to play a fundamental role in molecular recognition. Specifically, it is switch I which holds the key to discriminating between the two different G-interfaces. Moreover, residues which are characteristic for a given subgroup play subtle, but pivotal, roles in guaranteeing that the correct interfaces are formed.

HDX-MS reveals nucleotide-dependent, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon.

The bacterial Sec translocon is a multi-protein complex responsible for translocating diverse proteins across the plasma membrane. For post-translational protein translocation, the Sec-channel - SecYEG - associates with the motor protein SecA to mediate the ATP-dependent transport of pre-proteins across the membrane. Previously, a diffusional-based Brownian ratchet mechanism for protein secretion has been proposed; the structural dynamics required to facilitate this mechanism remain unknown. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel from Escherichia coli. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, and ATP-dependent, constriction of SecA around the pre-protein. ATP binding causes SecY to open and SecA to close; while, ADP produced by hydrolysis, has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery.

Nanometer-accuracy distance measurements between fluorophores at the single-molecule level

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil "stalk" of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

The Cryo-EM Structure and Activity of Kinesin-5 from Plasmodium falciparum: Mechanistic Lessons from a Parasite Kinesin

Kinesin superfamily members use ATP to drive mechanical output on the microtubule cytoskeleton. This mechanochemical behaviour has been specialised in the different kinesin families, and also between species. It has also been hypothesised that such differences could permit kinesin family, and even species specific inhibition by small-molecules. This triggered our structural and biochemical studies on the kinesin-5 family member from the malaria causing parasite – Plasmodium falciparum Kinesin-5 (PfK5). From this data we aimed to gain insight into the key aspects of kinesin mechanochemistry, and how this motor function may be adapted between species. In addition, we explored whether these adaptions can be exploited for the development of species specific inhibition of kinesin-5s - potentially opening the door to new anti-malarial therapeutics. To do this we employed single particle cryo-electron microscopy to visualize PfK5 in nucleotide-free and ATP-like stages of its mechanochemical cycle bound to microtubules. We used new techniques in 3D image processing and molecular modelling to obtain a near-atomic representation of PfK5's mechanochemical cycle. In conjunction with studies on PfK5's steady state ATP activity, and single molecule microtubule binding using total internal reflection microscopy, we have described PfK5 molecular properties. We find that PfK5 exhibits nucleotide dependent conformational changes and biochemical behaviour that are characteristic of a kinesin-5 family member. Conversely, we also observe evidence of some atypical biochemical properties which are explained by several unexpected conformations seen in our 3D reconstructions. Strikingly, a Plasmodium species specific insertion radically alters the chemical environment of the kinesin drug binding site, offering the possibility of PfK5-specific inhibition. Our results broaden our understanding of how evolution adapts the core function of eukaryotic kinesin mechanochemistry, and how we may utilise these differences in therapeutic development.

FtsA reshapes membrane architecture and remodels the Z-ring in Escherichia coli.

Cell division in prokaryotes initiates with assembly of the Z-ring at midcell, which, in Escherichia coli, is tethered to the inner leaflet of the cytoplasmic membrane through a direct interaction with FtsA, a widely conserved actin homolog. The Z-ring is comprised of polymers of tubulin-like FtsZ and has been suggested to provide the force for constriction. Here, we demonstrate that FtsA exerts force on membranes causing redistribution of membrane architecture, robustly hydrolyzes ATP and directly engages FtsZ polymers in a reconstituted system. Phospholipid reorganization by FtsA occurs rapidly and is mediated by insertion of a C-terminal membrane targeting sequence (MTS) into the bilayer and further promoted by a nucleotide-dependent conformational change relayed to the MTS. FtsA also recruits FtsZ to phospholipid vesicles via a direct interaction with the FtsZ C-terminus and regulates FtsZ assembly kinetics. These results implicate the actin homolog FtsA in establishment of a Z-ring scaffold, while directly remodeling the membrane and provide mechanistic insight into localized cell wall remodeling, invagination and constriction at the onset of division.

Conformational dynamics of the rotary subunit F in the A3 B3 DF complex of Methanosarcina mazei Gö1 A-ATP synthase monitored by single-molecule FRET.

In archaea the A1 AO ATP synthase uses a transmembrane electrochemical potential to generate ATP, while the soluble A1 domain (subunits A3 B3 DF) alone can hydrolyse ATP. The three nucleotide-binding AB pairs form a barrel-like structure with a central orifice that hosts the rotating central stalk subunits DF. ATP binding, hydrolysis and product release cause a conformational change inside the A:B-interface, which enforces the rotation of subunits DF. Recently, we reported that subunit F is a stimulator of ATPase activity. Here, we investigated the nucleotide-dependent conformational changes of subunit F relative to subunit D during ATP hydrolysis in the A3 B3 DF complex of the Methanosarcina mazei Gö1 A-ATP synthase using single-molecule Förster resonance energy transfer. We found two conformations for subunit F during ATP hydrolysis.

Architecture and Nucleotide-Dependent Conformational Changes of the Rvb1-Rvb2 AAA+ Complex Revealed by Cryoelectron Microscopy

Rvb1 and Rvb2 are essential AAA+ proteins that interact together during the assembly and activity of diverse macromolecules including chromatin remodelers INO80 and SWR-C, and ribonucleoprotein complexes including telomerase and snoRNPs. ATP hydrolysis by Rvb1/2 is required for function; however, the mechanism that drives substrate remodeling is unknown. Here we determined the architecture of the yeast Rvb1/2 dodecamer using cryoelectron microscopy and identify that the substrate-binding insertion domain undergoes conformational changes in response to nucleotide state. 2D and 3D classification defines the dodecamer flexibility, revealing distinct arrangements and the hexamer-hexamer interaction interface. Reconstructions of the apo, ATP, and ADP states identify that Rvb1/2 undergoes substantial conformational changes that include a twist in the insertion-domain position and a corresponding rotation of the AAA+ ring. These results reveal how the ATP hydrolysis cycle of the AAA+ domains directs insertion-domain movements that could provide mechanical force during remodeling or helicase activities.

Nucleotide-dependent conformational changes of the AAA+ ATPase p97 revisited.

The ubiquitous AAA-ATPase p97 segregates ubiquitylated proteins from their molecular environment. Previous studies of the nucleotide-dependent conformational changes of p97 were inconclusive. Here, we determined its structure in the presence of ADP, AMP-PNP, or ATP-γS at 6.1-7.4 Å resolution using single particle cryo-electron microscopy. Both AAA domains, D1 and D2, assemble into essentially six-fold symmetrical rings. The pore of the D1-ring remains essentially closed under all nucleotide conditions, whereas the D2-ring shows an iris-like opening for ADP. The largest conformational changes of p97 are 'swinging motions' of the N-terminal domains, which may enable segregation of ubiquitylated substrates from their environment.

Novel topography of the Rab11-effector interaction network within a ciliary membrane targeting complex

Small GTPases function as universal molecular switches due to the nucleotide dependent conformational changes of their switch regions that allow interacting proteins to discriminate between the active GTP-bound and the inactive GDP-bound states. Guanine nucleotide exchange factors (GEFs) recognize the inactive GDP-bound conformation whereas GTPase activating proteins (GAPs), and the GTPase effectors recognize the active GTP-bound state. Small GTPases are linked to each other through regulatory and effector proteins into functional networks that regulate intracellular membrane traffic through diverse mechanisms that include GEF and GAP cascades, GEF-effector interactions, common effectors and positive feedback loops linking interacting proteins. As more structural and functional information is becoming available, new types of interactions between regulatory proteins, and new mechanisms by which GTPases are networked to control membrane traffic are being revealed. This review will focus on the structure and function of the novel Rab11-FIP3-Rabin8 dual effector complex and its implications for the targeting of sensory receptors to primary cilia, dysfunction of which causes cilia defects underlying human diseases and disorders know as ciliopathies.

Structure and mechanism of the ATPase that powers viral genome packaging

Many viruses package their genomes into procapsids using an ATPase machine that is among the most powerful known biological motors. However, how this motor couples ATP hydrolysis to DNA translocation is still unknown. Here, we introduce a model system with unique properties for studying motor structure and mechanism. We describe crystal structures of the packaging motor ATPase domain that exhibit nucleotide-dependent conformational changes involving a large rotation of an entire subdomain. We also identify the arginine finger residue that catalyzes ATP hydrolysis in a neighboring motor subunit, illustrating that previous models for motor structure need revision. Our findings allow us to derive a structural model for the motor ring, which we validate using small-angle X-ray scattering and comparisons with previously published data. We illustrate the model's predictive power by identifying the motor's DNA-binding and assembly motifs. Finally, we integrate our results to propose a mechanistic model for DNA translocation by this molecular machine.

Histidine 114 Is Critical for ATP Hydrolysis by the Universally Conserved ATPase YchF

GTPases perform a wide range of functions, ranging from protein synthesis to cell signaling. Of all known GTPases, only eight are conserved across all three domains of life. YchF is one of these eight universally conserved GTPases; however, its cellular function and enzymatic properties are poorly understood. YchF differs from the classical GTPases in that it has a higher affinity for ATP than for GTP and is a functional ATPase. As a hydrophobic amino acid-substituted ATPase, YchF does not possess the canonical catalytic Gln required for nucleotide hydrolysis. To elucidate the catalytic mechanism of ATP hydrolysis by YchF, we have taken a two-pronged approach combining classical biochemical and in silico techniques. The use of molecular dynamics simulations allowed us to complement our biochemical findings with information about the structural dynamics of YchF. We have thereby identified the highly conserved His-114 as critical for the ATPase activity of YchF from Escherichia coli. His-114 is located in a flexible loop of the G-domain, which undergoes nucleotide-dependent conformational changes. The use of a catalytic His is also observed in the hydrophobic amino acid-substituted GTPase RbgA and is an identifier of the translational GTPase family.

A Nucleotide-Driven Switch Regulates Flanking DNA Length Sensing by a Dimeric Chromatin Remodeler

The ATP-dependent chromatin assembly factor (ACF) spaces nucleosomes to promote formation of silent chromatin. Two copies of its ATPase subunit SNF2h bind opposite sides of a nucleosome, but how these protomers avoid competition is unknown. SNF2h senses the length of DNA flanking a nucleosome via its HAND-SANT-SLIDE (HSS) domain, yet it is unclear how this interaction enhances remodeling. Using covalently connected SNF2h dimers we show that dimerization accelerates remodeling and that the HSS contributes to communication between protomers. We further identify a nucleotide-dependent conformational change in SNF2h. In one conformation the HSS binds flanking DNA, and in another conformation the HSS engages the nucleosome core. Based on these results, we propose a model in which DNA length sensing and translocation are performed by two distinct conformational states of SNF2h. Such separation of function suggests that these activities could be independently regulated to affect remodeling outcomes.

A Structural Model of the Kinesin-5 Mechanochemical Cycle

Kinesin motor domains drive diverse microtubule-based, ATP-dependent activities but the molecular adaptations that specify these diverse functions are poorly understood. Kinesin-5s are essential mitotic motors and their inhibition with specific small molecules blocks cell division. Using cryo-electron microscopy and subnanometer resolution structure determination, we have visualised conformations of microtubule-bound human kinesin-5 motor domain at successive steps in its ATPase cycle. In the ATP-like state, the kinesin-5 neck-linker is directed towards the microtubule plus-end, consistent with its role in directional force generation. As ATP hydrolysis proceeds, nucleotide-dependent conformational changes in the active site are allosterically propagated into rotations of the motor domain, uncurling of the drug-binding loop5 and discrete, ratchet-like displacements of the neck linker that contribute to motor stepping. The motor N-terminus also undergoes large reorientations that indicate its role in controlling kinesin-5 neck-linker conformation throughout the motor's ATPase cycle. A kinesin-5 mutant lacking this N-terminus is enzymatically active, but ATP-dependent neck linker movement and motility is defective, although not totally ablated. Our data demonstrate that, while the motor N-terminus plays a kinetic role in controlling efficient neck-linker movement, the kinesin-5 neck-linker has intrinsic biophysical properties that enable it undergo nucleotide-dependent ratchet-like movements that have presumably evolved according to specific functional requirements.

A Mutation in the N Domain of Escherichia coli Lon Stabilizes Dodecamers and Selectively Alters Degradation of Model Substrates

Escherichia coli Lon, an ATP-dependent AAA(+) protease, recognizes and degrades many different substrates, including the RcsA and SulA regulatory proteins. More than a decade ago, the E240K mutation in the N domain of Lon was shown to prevent degradation of RcsA but not SulA in vivo. Here, we characterize the biochemical properties of the E240K mutant in vitro and present evidence that the effects of this mutation are complex. For example, Lon(E240K) exists almost exclusively as a dodecamer, whereas wild-type Lon equilibrates between hexamers and dodecamers. Moreover, Lon(E240K) displays degradation defects in vitro that do not correlate in any simple fashion with degron identity, substrate stability, or dodecamer formation. The Lon sequence segment near residue 240 is known to undergo nucleotide-dependent conformational changes, and our results suggest that this region may be important for coupling substrate binding with allosteric activation of Lon protease and ATPase activity.

Molecular Disruption of the Power Stroke in the ATP-binding Cassette Transport Protein MsbA

ATP-binding cassette transporters affect drug pharmacokinetics and are associated with inherited human diseases and impaired chemotherapeutic treatment of cancers and microbial infections. Current alternating access models for ATP-binding cassette exporter activity suggest that ATP binding at the two cytosolic nucleotide-binding domains provides a power stroke for the conformational switch of the two membrane domains from the inward-facing conformation to the outward-facing conformation. In outward-facing crystal structures of the bacterial homodimeric ATP-binding cassette transporters MsbA from gram-negative bacteria and Sav1866 from Staphylococcus aureus, two transmembrane helices (3 and 4) in the membrane domains have their cytoplasmic extensions in close proximity, forming a tetrahelix bundle interface. In biochemical experiments on MsbA from Escherichia coli, we show for the first time that a robust network of inter-monomer interactions in the tetrahelix bundle is crucial for the transmission of nucleotide-dependent conformational changes to the extracellular side of the membrane domains. Our observations are the first to suggest that modulation of tetrahelix bundle interactions in ATP-binding cassette exporters might offer a potent strategy to alter their transport activity.

The Structural Basis of Force Generation by the Mitotic Motor Kinesin-5

Kinesin-5s are essential for forming the bipolar spindle during mitosis in most eukaryotes. The kinesin-5 motor domain contains conserved nucleotide and microtubule binding sites and mechanical elements to generate force. However, biochemical and biophysical studies have suggested that the mechano-chemistry of the kinesin-5 motor is very different from other kinesins. using cryo-electron microscopy and image reconstruction, we have calculated sub-nanometer resolution structures of microtubule-bound human kinesin-5 before and after nucleotide binding. Our structures reveal that, despite its mechanistic differences with conventional kinesin, kinesin-5 has the same coupled, nucleotide-dependent conformational changes as seen in conventional kinesins, including a ratchet-like docking of the neck linker, and simultaneous, parallel docking of the amino-terminal cover strand. These observations are supported by kinetic experiments that indicate a cooperative rearrangement of the kinesin-5-specific neck linker with the amino-terminal cover strand during the motor's ATPase cycle. In contrast to conventional kinesin however, our structures reveal a dramatic reorientation of Loop L5 - the binding site for allosteric inhibitors of kinesin-5-following ATP binding. This reorientation suggests that L5 is directly involved in controlling nucleotide binding by acting as an intra-molecular competitive inhibitor. Our structures indicate that allosteric inhibitors of human kinesin-5 bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Our data reveal the structural basis for kinesin-5 force generation that has evolved in the physiological context of the mitotic spindle.

Building a fission machine – structural insights into dynamin assembly and activation

Dynamin is a large multidomain GTPase that assembles into helical arrays around the necks of deeply invaginated clathrin-coated pits and catalyzes membrane fission during the final stages of endocytosis. Although it is well established that the function of dynamin in vivo depends on its oligomerization and its capacity for efficient GTP hydrolysis, the molecular mechanisms governing these activities have remained poorly defined. In recent years, there has been an explosion of structural data that has provided new insights into the architecture, organization and nucleotide-dependent conformational changes of the dynamin fission machine. Here, we review the key findings of these efforts and discuss the implications of each with regard to GTP hydrolysis, dynamin assembly and membrane fission.