Domain Conformational Changes Research Articles

Unraveling the Interplay of Extracellular Domain Conformational Changes and Parathyroid Hormone Type 1 Receptor Activation in Class B1 G Protein-Coupled Receptors: Integrating Enhanced Sampling Molecular Dynamics Simulations and Markov State Models.

Parathyroid hormone (PTH) type 1 receptor (PTH1R), as a typical class B1 G protein-coupled receptor (GPCR), is responsible for regulating bone turnover and maintaining calcium homeostasis, and its dysregulation has been implicated in the development of several diseases. The extracellular domain (ECD) of PTH1R is crucial for the recognition and binding of ligands, and the receptor may exhibit an autoinhibited state with the closure of the ECD in the absence of ligands. However, the correlation between ECD conformations and PTH1R activation remains unclear. Thus, this study combines enhanced sampling molecular dynamics (MD) simulations and Markov state models (MSMs) to reveal the possible relevance between the ECD conformations and the activation of PTH1R. First, 22 intermediate structures are generated from the autoinhibited state to the active state and conducted for 10 independent 200 ns simulations each. Then, the MSM is constructed based on the cumulative 44 μs simulations with six identified microstates. Finally, the potential interplay between ECD conformational changes and PTH1R activation as well as cryptic allosteric pockets in the intermediate states during receptor activation is revealed. Overall, our findings reveal that the activation of PTH1R has a specific correlation with ECD conformational changes and provide essential insights for GPCR biology and developing novel allosteric modulators targeting cryptic sites.

A SLiM-dependent conformational change in baculovirus IE1 controls its focus formation ability.

The baculovirus IE1 gene encodes a multifunctional protein that is essential for both DNA replication and RNA transcription of the virus. Prior to viral DNA replication, IE1 promotes early gene transcription when localized in hr-dependent foci. During viral DNA replication, the IE1 foci expand and fuse to generate the virogenic stroma (VS) with IE1 found in the VS reticulum. To explore the IE1 structural features essential for this coordinated localization, we constructed various IE1 mutants based on three putative domains (N, I, and C). We determined that a BDI motif located in the intrinsic disorder region (IDR) between the N and I domains acts as a nuclear localization signal, whereas BDII and HLH in the C domain are required for VS localization in infected cells or for chromosomal association in uninfected mitotic cells. Deletion of the SLiM (short linear motif) located in the I domain restrains both nuclear- and VS localization. Intra-molecular fluorescence resonance energy transfer (FRET) probes of IE1 mutants revealed a conformational change of the I-C two-domain fragment during infection, which was inhibited by aphidicolin, suggesting that IE1 undergoes a stage-dependent conformational change. Further, homo-dimerization of the I domain and stage-dependent conformational changes require an intact SLiM. Mutational analysis of SLiM revealed that VS localization and chromosomal association were retained following S291A and S291E substitutions, but hr-dependent focus formation differed between the two mutations. These results suggest that coordinated IE1 localization is controlled by SLiM-dependent conformational changes that are potentially switched by the phosphorylation state of the SLiM.

Focused classifications and refinements in high-resolution single particle cryo-EM analysis

Recent advances in cryo electron microscopy (cryo-EM) and image processing provide new opportunities to analyse drug targets at high resolution. However, structural heterogeneity limits resolution in many practical cases, hence restricting the level at which structural details can be analysed and drug design be performed. As structural disorder is not spread throughout the entire structure of a given macromolecular complex but instead is found in certain regions that move with respect to others and covering molecular scales from domain conformational changes up to the level of side chain conformations in ligand binding pockets, it is possible to focus the attention on those regions and the associated relative movements. Here we show how the usage of focused classifications and refinements provide insights into global conformational arrangements, exemplified on the human ribosome and on the cannabinoid G protein coupled receptor (GPCR), and how they can improve the local map resolution from an essentially disordered region to the 3–4 Å and finally to the 2 Å resolution range. A systematic analysis with variable spherical masks during focused refinement is presented showing that the choice of an optimal mask size helps refining to high resolution. This study covers several practical approaches on 4 examples illustrating how important mask size & shape and including neighbouring structural elements are for a focused analysis of a macromolecular complex. Such methods will be crucial for cryo-EM structure-based drug design of various medical targets and are applicable to single particle cryo-EM and electron tomography data.

Intramolecular tension and catch bonds play key roles in the conformational cycle of the dynein-like AAA mechanoenzyme Mdn1.

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically extract the assembly factors Rsa4 and Ytm1 from the pre-60S subunit during ribosome biogenesis. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with various activities) ring, which like dynein has six ATPase sites. The AAA ring is followed by large (>2000 amino acid) linking domains including an ∼500 amino acid disordered (D/E-rich) region, and a C-terminal assembly factor-binding MIDAS domain. Previous structural studies have revealed that when Mdn1 binds to the pre-60S, it docks its MIDAS domain onto its AAA ring and forms a tripartite connection between these two domains and the assembly factor that it is primed to extract. However, it is currently not known how this conformational state is formed and regulated. Here, we use a combination of optical tweezers, bulk biochemistry, and chemical biology approaches to investigate domain interactions and conformational changes in the Mdn1 mechanochemical cycle. We find that the MIDAS domain can bind stably to the AAA ring when they are separated into two different proteins, but that intramolecular tension built up along the linking domains prohibits this interaction in the full-length protein. We further find that the MIDAS domain can form catch bonds - bonds where tension enhances binding affinity - with both Rsa4 and Ytm1. Together, these results suggest a mechanoregulatory mechanism wherein intramolecular tension prevents MIDAS-AAA docking when Mdn1 is off the pre-60S, yet promotes the docked conformation when Mdn1 is on the pre-60S by activating the catch bond.

FSHR activation through small molecule modulators: Mechanistic insights from MD simulations

Follicle-stimulating hormone receptor (FSHR) is a glycoprotein hormone receptor that plays a vital role in reproduction, cancer progression and osteoporosis. Owing to its therapeutic importance, several small molecule modulators have been identified by researchers through high throughput studies that usually include virtual screening of chemical libraries followed by in vitro validation through radio-ligand binding assays, cAMP accumulation and luciferase-based luminescence assays. The binding site of these modulators and structural changes that accompany modulator binding remains elusive. Here, we address these aspects through molecular docking and MD simulations on well-studied FSHR modulators and comparing the domain motions between agonist/FSH bound and antagonist bound FSHR structures. It was observed that agonist and antagonist modulators bind to the same site, but interact with distinct residues in transmembrane domain(TMD). FSHR(TMD) residues Ile522, Ala595, Ile602 and Val604 were found to interact only with agonist. Notably, these residues are conserved in the close homolog luteinizing hormone/choriogonadotropin receptor (LHCGR) and participate in interaction with its agonist Org43553. We observed distinctly prominent domain motions and conformational changes in TM helices 3, 4 and 6 for agonist bound FSHR structure. These structural changes have also been reported for LHCGR, and few GPCR members suggesting an important and well conserved mechanism of GPHR activation that could be exploited for design of novel modulators.

Multimodal approach to characterize the tetrameric form of human PML-RBCC domain and ATO-mediated conformational changes

RING-B box-coiled coil (RBCC) domain of promyelocytic leukemia (PML) comprises a zinc finger motif that is targeted by arsenic trioxide (ATO) to treat acute promyelocytic leukemia (APL) pathogenesis. Preliminary evidence suggests that the PML-RBCC has different functional characteristics, but no structural details have been reported despite its importance in differential expression and cell-cycle regulation. Therefore, the recombinant h-PML-RBCC protein was purified to its homogeneity, and characterized for oligomeric behaviour which indicated that RBCC domain exists as a tetramer in solution. Furthermore, nano-DSF and circular-dichroism demonstrated that the tetrameric form preserves its native conformation along with thermal stability (Tm = 83.2 °C). In-silico-based PML-RBCC structure was used to perform the molecular dynamics simulation for 300 ns in the presence of zinc atoms, which demonstrated the differential dynamic of PML-RBCC tetrameric chains. MMPBSA analysis also indicated the role of hydrophobic interactions that favours stable tetrameric structure of PML-RBCC. ATO-induced secondary and tertiary structure changes were observed in PML-RBCC using circular dichroism and fluorescence spectroscopy. Dynamic light scattering and transmission electron microscopy revealed ATO-induced higher-order oligomerization and aggregation of PML-RBCC. The unique oligomeric nature of the h-PML-RBCC protein and its interactions with ATO will help to understand the mechanism of APL pathogenesis and drug resistance.

Discrepancy in interactions and conformational dynamics of pregnane X receptor (PXR) bound to an agonist and a novel competitive antagonist

Pregnane X receptor (PXR) is a nuclear receptor with an essential role in regulating drug metabolism genes. While the mechanism of action for ligand-mediated PXR agonism is well-examined, its ligand-mediated inhibition or antagonism is poorly understood. Here we employ microsecond timescale all-atom molecular dynamics (MD) simulations to investigate how our newly identified dual kinase and PXR inhibitor, compound 100, acts as a competitive PXR antagonist and not as a full agonist. We study the PXR ligand binding domain conformational changes associated with compound 100 and compare the results to the full agonist SR12813, in presence and absence of the coactivator. Furthermore, we complement our research by experimentally disclosing the effect of eight key-residue mutations on PXR activation. Finally, simulations of P2X4 inhibitor (BAY-1797) in complex with PXR, which shares an identical structural moiety with compound 100, provide further insights to ligand-induced PXR behaviour. Our MD data suggests ligand-specific influence on conformations of different PXR-LBD regions, including α6 region, αAF-2, α1-α2′, β1′-α3 and β1-β1′ loop. Our results provide important insights on conformational behaviour of PXR and offers guidance how to alleviate PXR agonism or to promote PXR antagonism.

Structural Studies on the Shapeshifting Murine Norovirus.

Noroviruses are responsible for almost a fifth of all cases of gastroenteritis worldwide. The calicivirus capsid is composed of 180 copies of VP1 with a molecular weight of ~58 kDa. This coat protein is divided into the N-terminus (N), the shell (S) and C-terminal protruding (P) domains. The S domain forms a shell around the viral RNA genome, while the P domains dimerize to form protrusions on the capsid surface. The P domain is subdivided into P1 and P2 subdomains, with the latter containing the binding sites for cellular receptors and neutralizing antibodies. Reviewed here are studies on murine norovirus (MNV) showing that the capsid responds to several physiologically relevant cues; bile, pH, Mg2+, and Ca2+. In the initial site of infection, the intestinal tract, high bile and metal concentrations and low pH cause two significant conformational changes: (1) the P domain contracts onto the shell domain and (2) several conformational changes within the P domain lead to enhanced receptor binding while blocking antibody neutralization. In contrast, the pH is neutral, and the concentrations of bile and metals are low in the serum. Under these conditions, the loops at the tip of the P domain are in the open conformation with the P domain floating on a linker or tether above the shell. This conformational state favors antibody binding but reduces interactions with the receptor. In this way, MNV uses metabolites and environmental cues in the intestine to optimize cellular attachment and escape antibody binding but presents a wholly different structure to the immune system in the serum. To our knowledge, this is the first example of a virus shapeshifting in this manner to escape the immune response.

Structures of heat shock factor trimers bound to DNA

SummaryHeat shock factor 1 (HSF1) and 2 (HSF2) play distinct but overlapping regulatory roles in maintaining cellular proteostasis or mediating cell differentiation and development. Upon activation, both HSFs trimerize and bind to heat shock elements (HSEs) present in the promoter region of target genes. Despite structural insights gained from recent studies, structures reflecting the physiological architecture of this transcriptional machinery remains to be determined. Here, we present co-crystal structures of human HSF1 and HSF2 trimers bound to DNA, which reveal a triangular arrangement of the three DNA-binding domains (DBDs) with protein-protein interactions largely mediated by the wing domain. Two structural properties, different flexibility of the wing domain and local DNA conformational changes induced by HSF binding, seem likely to contribute to the subtle differential specificity between HSF1 and HSF2. Besides, two more structures showing DBDs bound to “two-site” head-to-head HSEs were determined as additions to the published tail-to-tail dimer-binding structures.

Emerging roles of adhesion G protein-coupled receptors.

Adhesion G protein-coupled receptors (aGPCRs) form a sub-group within the GPCR superfamily. Their distinctive structure contains an abnormally large N-terminal, extracellular region with a GPCR autoproteolysis-inducing (GAIN) domain. In most aGPCRs, the GAIN domain constitutively cleaves the receptor into two fragments. This process is often required for aGPCR signalling. Over the last two decades, much research has focussed on aGPCR-ligand interactions, in an attempt to deorphanize the family. Most ligands have been found to bind to regions N-terminal to the GAIN domain. These receptors may bind a variety of ligands, ranging across membrane-bound proteins and extracellular matrix components. Recent advancements have revealed a conserved method of aGPCR activation involving a tethered ligand within the GAIN domain. Evidence for this comes from increased activity in receptor mutants exposing the tethered ligand. As a result, G protein-coupling partners of aGPCRs have been more extensively characterised, making use of their tethered ligand to create constitutively active mutants. This has led to demonstrations of aGPCR function in, for example, neurodevelopment and tumour growth. However, questions remain around the ligands that may bind many aGPCRs, how this binding is translated into changes in the GAIN domain, and the exact mechanism of aGPCR activation following GAIN domain conformational changes. This review aims to examine the current knowledge around aGPCR activation, including ligand binding sites, the mechanism of GAIN domain-mediated receptor activation and how aGPCR transmembrane domains may relate to activation. Other aspects of aGPCR signalling will be touched upon, such as downstream effectors and physiological roles.

Structures of Heat Shock Factor Trimers Bound to DNA Reveal the Panoramic Architecture of the HSF-Regulated Transcriptional Machinery

Heat shock factor 1 (HSF1) and 2 (HSF2) play distinct but overlapping regulatory roles in maintaining cellular protein homeostasis or mediating cell differentiation anddevelopment. Upon activation, both HSFs trimerize and bind to heat shock elements(HSEs) present in the promoter region of target genes. Despite innovative insights gained from recent structural studies, the panoramic architecture of this transcriptional machinery remains to be determined. Here, we present crystal structures of humanHSF1 and HSF2 trimers bound to DNA, which reveal a triangular arrangement of thethree DNA-binding domains (DBDs) with protein-protein interactions largely mediated by the wing domain. Two structural properties, the different flexibility of the wing domain and local DNA conformational changes induced by HSF binding, seem likelyto contribute to the subtle differential specificity between HSF1 and HSF2. Besides, two more structures showing DBDs bound to ‘two-site’ head-to-head HSEs weredetermined as additions to the published tail-to-tail dimer-binding structures. Our structures lay a foundation for further mechanistic study on how HSFs fulfill complex regulation on different target genes.

ATP Hydrolysis by the SNF2 Domain of Dnmt5 Is Coupled to Both Specific Recognition and Modification of Hemimethylated DNA

C.neoformans Dnmt5 is an unusually specific maintenance-type CpG methyltransferase (DNMT) that mediates long-term epigenome evolution. It harbors a DNMT domain and SNF2 ATPase domain. We find that the SNF2 domain couples substrate specificity to an ATPase step essential for DNA methylation. Coupling occurs independent of nucleosomes. Hemimethylated DNA preferentially stimulates ATPase activity, and mutating Dnmt5's ATP-binding pocket disproportionately reduces ATPase stimulation by hemimethylated versus unmethylated substrates. Engineered DNA substrates that stabilize a reaction intermediate by mimicking a "flipped-out" conformation of the target cytosine bypass the SNF2 domain's requirement for hemimethylation. This result implies that ATP hydrolysis by the SNF2 domain is coupled to the DNMT domain conformational changes induced by preferred substrates. These findings establish a new role for a SNF2 ATPase: controlling an adjoined enzymatic domain's substrate recognition and catalysis. We speculate that this coupling contributes to the exquisite specificity of Dnmt5 via mechanisms related to kinetic proofreading.

Nudged Elastic Band Study on the N-Terminal Domain Conformational Pathways of Annexin A1 from a Buried State to an Exposed State.

Mapping the conformational pathway of biomolecules is a great challenge because of the large size and complexity of biomolecules. The nudged elastic band (NEB) method has been applied to study the reaction pathways for both small organic molecules and small peptides of a few amino acids. In this work, for the first time, the NEB method was employed to study the conformational pathways of Annexin A1, a membrane-binding protein of 334 amino acids. The N-terminal domain conformational change from the buried state within the core domain to the exposed state outside the core domain is a vital step for Annexin A1 to interact with membranes or target proteins. In this work, multiple molecular dynamics simulations using the NEB method were performed to simulate the N-terminal domain conformational pathway of Annexin A1. Our results suggested that the N-terminal domain of Annexin A1 is removed from the repeat III of the core domain in a sliding motion. The loop region of repeat III covering the N-terminal helix in the buried state does not lift up for the N-terminal to swing out of the pocket; instead, the N-terminal pulls out from the bottom of the core domain. The N-terminal domain linker region (S27-N42) flexibility is critical for the N-terminal domain conformational changes. Our results also suggested a two-step folding process for the helix D in repeat III, M247-V250 folds first followed by the folding of L251-E254. The results demonstrated that the NEB method could be an effective tool for theoretical studies on conformational pathways of biomolecules.

Timing Is Everything: Impact of Naturally Occurring Staphylococcus aureus AgrC Cytoplasmic Domain Adaptive Mutations on Autoinduction.

Mutations in the polymorphic Staphylococcus aureusagr locus responsible for quorum sensing (QS)-dependent virulence gene regulation occur frequently during host adaptation. In two genomically closely related S. aureus clinical isolates exhibiting marked differences in Panton-Valentine leukocidin production, a mutation conferring an N267I substitution was identified in the cytoplasmic domain of the QS sensor kinase, AgrC. This natural mutation delayed the onset and accumulation of autoinducing peptide (AIP) and showed reduced responsiveness to exogenous AIPs. Other S. aureus strains harboring naturally occurring AgrC cytoplasmic domain mutations were identified, including T247I, I311T, A343T, L245S, and F264C. These mutations were associated with reduced cytotoxicity, delayed/reduced AIP production, and impaired sensitivity to exogenous AIP. Molecular dynamics simulations were used to model the AgrC cytoplasmic domain conformational changes arising. Although mutations were localized in different parts of the C-terminal domain, their impact on molecular structure was manifested by twisting of the leading helical hairpin α1-α2, accompanied by repositioning of the H-box and G-box, along with closure of the flexible loop connecting the two and occlusion of the ATP-binding site. Such conformational rearrangements of key functional subdomains in these mutants highlight the cooperative response of molecular structure involving dimerization and ATP binding and phosphorylation, as well as the binding site for the downstream response element AgrA. These appear to increase the threshold for agr activation via AIP-dependent autoinduction, thus reducing virulence and maintaining S. aureus in an agr-downregulated "colonization" mode.IMPORTANCE Virulence factor expression in Staphylococcus aureus is regulated via autoinducing peptide (AIP)-dependent activation of the sensor kinase AgrC, which forms an integral part of the agr quorum sensing system. In response to bound AIP, the cytoplasmic domain of AgrC (AgrC-cyt) undergoes conformational changes resulting in dimerization, autophosphorylation, and phosphotransfer to the response regulator AgrA. Naturally occurring mutations in AgrC-cyt are consistent with repositioning of key functional domains, impairing dimerization and restricting access to the ATP-binding pocket. Strains harboring specific AgrC-cyt mutations exhibit reduced AIP autoinduction efficiency and a timing-dependent attenuation of cytotoxicity which may confer a survival advantage during established infection by promoting colonization while restricting unnecessary overproduction of exotoxins.

Cryo-electron microscopy structures of ArnA, a key enzyme for polymyxin resistance, revealed unexpected oligomerizations and domain movements

Gram-negative bacteria evade the attack of cationic antimicrobial peptides through modifying their lipid A structure in their outer membranes with 4-amino-4-deoxy-L-arabinose (Ara4N). ArnA is a crucial enzyme in the lipid A modification pathway and its deletion abolishes the polymyxin resistance of gram-negative bacteria. Previous studies by X-ray crystallography have shown that full-length ArnA forms a three-bladed propeller-shaped hexamer. Here, the structures of ArnA determined by cryo-electron microscopy (cryo-EM) reveal that ArnA exists in two 3D architectures, hexamer and tetramer. This is the first observation of a tetrameric ArnA. The hexameric cryo-EM structure is similar to previous crystal structures but shows differences in domain movements and conformational changes. We propose that ArnA oligomeric states are in a dynamic equilibrium, where the hexamer state is energetically more favorable, and its domain movements are important for cooperating with downstream enzymes in the lipid A-Ara4N modification pathway. The results provide us with new possibilities to explore inhibitors targeting ArnA.

Characterization of the Assembly Dynamics of Streptococcus Pneumoniae FtsZ using Intrinsic Tryptophan Fluorescence

FtsZ is a prokaryotic cytoskeletal protein, which assembles into the Z‐ring at the division site and orchestrates the cell division process in bacteria. FtsZ has GTPase activity and self‐assembles to form polymers. We investigated the assembly characteristics of FtsZ from the pathogenic organism, Streptococcus pneumoniae. The critical concentration for FtsZ polymerization was found to be 2.4 ± 0.1 and 2.2 ± 0.2 μM measured using sedimentation assay and GTPase assay, respectively. The results indicated that the GTPase activity and assembly are closely linked to each other. We identified that FtsZ from Streptococcus species contains two native tryptophan (Trp) residues unique to this genus. Interestingly, we observed that during polymerization, the tryptophan fluorescence of FtsZ increases showing that the assembly of FtsZ can be monitored using the native tryptophan fluorescence. Using tryptophan fluorescence along with light scattering, sedimentation assay and electron microscopy we examined the effects of divalent cations, calcium and magnesium, on FtsZ assembly. Divalent magnesium enhanced the tryptophan fluorescence change during FtsZ assembly showing that it promoted the longitudinal assembly of FtsZ. Divalent calcium induced the bundling of FtsZ filaments thereby affecting the lateral interactions of FtsZ polymers but did not enhance the tryptophan fluorescence, showing the sensitivity of tryptophan fluorescence to longitudinal assembly. Next, we sought to investigate the contribution of individual tryptophan residues to fluorescence of FtsZ and use them for understanding the assembly of FtsZ. A homology model for FtsZ from Streptococcus pneumoniae showed that the two tryptophan residues were present at the C‐terminal of the protein – one at the 298th position of FtsZ near the T7 loop and the other at 378th position in the C‐terminal tail region. We used site‐directed mutagenesis to introduce point mutations to FtsZ at these positions to mutate individual tryptophan residues to cysteine or phenylalanine residues to create W294C/F and W378C/F mutant proteins. W378C/F mutants showed a similar change in tryptophan fluorescence during polymerization as of wild‐type, while W294C/F mutants did not show any change. This indicated that the Trp at the 294th position is responsible for the change in fluorescence during polymerization. This further suggested that the environment around 294W changes during the polymerization while the local environment around the 378W does not change. The two tryptophan residues in the protein allow us to use the intrinsic tryptophan fluorescence to gain new insights into FtsZ assembly. Further, it would be interesting to explore the use of these mutant proteins in following the domain movements and conformational changes occurring during the assembly and disassembly of FtsZ.Support or Funding InformationThe work is supported by a grant from Department of Science and Technology (DST, India) to D.P.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Conformational Dynamics, Intramolecular Domain Conformation Signaling, and Activation of Apo-FimD Revealed by Single-Molecule Fluorescence Resonance Energy Transfer Studies.

The chaperone-usher secretion pathway is a conserved bacterial protein secretion system dedicated to the biogenesis of adhesive fibers. Usher, a multidomain-containing outer membrane protein, plays a central role in this process by acting as a molecular machine that recruits different chaperone-subunit complexes, catalyzes subunit polymerization, and forms a channel for secretion of the assembled subunits. While recent crystal structural studies have greatly advanced our understanding of the structure and function of ushers, the overall architecture of the full-length apo-usher, the molecular events that dictate conformational changes in usher during pilus biogenesis, and its activation by the specific chaperone-adhesin complex remain largely elusive. Using single-molecule fluorescence resonance energy transfer studies, we found that the substrate-free usher FimD (apo-FimD) adopts a contracted conformation that is distinct from its substrate-bound states; both the N-terminal domain (NTD) and the C-terminal domain (CTD) of apo-FimD are highly dynamic, and FimD coordinates its domain conformational changes via intramolecular domain conformation signaling. By combining these studies with in vitro photo-cross-linking studies, we further show that only the chaperone-bound adhesin (FimC:FimH) can be transferred to the CTD, dislocates the plug domain, and triggers conformational changes in the remaining FimD domains. Taken together, these studies delineate an overall architecture of the full-length apo-FimD, provide detailed mechanic insight into the activation of apo-FimD, and explain why FimD could adjust its conformational states to perform multiple functions in each cycle of pilus subunit addition and ensure that pilus assembly proceeds progressively in a cellular energy-free environment.

Structure of an EIIC sugar transporter trapped in an inward-facing conformation

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) transports sugar into bacteria and phosphorylates the sugar for metabolic consumption. The PTS is important for the survival of bacteria and thus a potential target for antibiotics, but its mechanism of sugar uptake and phosphorylation remains unclear. The PTS is composed of multiple proteins, and the membrane-embedded Enzyme IIC (EIIC) component transports sugars across the membrane. Crystal structures of two members of the glucose superfamily of EIICs, bcChbC and bcMalT, were solved in the inward-facing and outward-facing conformations, and the structures suggest that sugar translocation could be achieved by movement of a structured domain that contains the sugar-binding site. However, different conformations have not been captured on the same transporter to allow precise description of the conformational changes. Here we present a crystal structure of bcMalT trapped in an inward-facing conformation by a mercury ion that bridges two strategically placed cysteine residues. The structure allows direct comparison of the outward- and inward-facing conformations and reveals a large rigid-body motion of the sugar-binding domain and other conformational changes that accompany the rigid-body motion. All-atom molecular dynamics simulations show that the inward-facing structure is stable with or without the cross-linking. The conformational changes were further validated by single-molecule Föster resonance energy transfer (smFRET). Combined, these results establish the elevator-type mechanism of transport in the glucose superfamily of EIIC transporters.

Domain Formation and Conformational Changes in Gold Nanoparticle Conjugates Studied Using DPD Simulations.

A gold nanoparticle (AuNP) conjugate formed with 11-mercaptoundecanoic acid (MUA) and thiolated polyethylene glycol (SH-PEG) is simulated using dissipative particle dynamics (DPD) methods, obtaining an excellent agreement with previous experimental observations. The simulations cover the isolated components (AuNP, MUA, and SH-PEG), as well as pairs of components, and finally the all three components at the same time. In this latter case, changes in the order of addition of MUA and SH-PEG over the AuNP are also considered. The AuNP is formed by independent gold beads and keeps an almost spherical shape throughout the simulation. MUA forms micelles of four to six MUA units when dispersed in water, while SH-PEG stays individually and well solvated. When exposed to AuNP, both molecules show a tendency to form patches on the surface. SH-PEG displays two different conformations (radial and tangential) depending on its relative concentration and the presence of other molecules at the NP surface. When combined at subsaturation concentrations, MUA arrives faster to the AuNP surface than SH-PEG and forms patches while SH-PEG occupies the remaining free surface. In these conditions, the order of addition of the different components partially alters these results. When SH-PEG is added over an already formed MUA/AuNP partial layer, it adopts a radial conformation over the MUA formed patches; on the contrary, if MUA is added over an already formed SH-PEG/AuNP partial layer, much less SH-PEGs adopt a radial conformation and MUA patches are significantly smaller.

Crystal Structure and Biochemical Characterization of an Aminopeptidase LapB from Legionella pneumophila.

Aminopeptidases are a group of exopeptidases that catalyze the removal of a wide range of N-terminal amino acid residues from peptides and proteins. They have many important commercial applications in the food industry. We determined the crystal structure of an aminopeptidase LapB from Legionella pneumophila. The overall structure reveals that the N-terminal protease-associated (PA) domain presents a new fold and shields the active site cavity of the conserved C-terminal peptidase domain. The steady-state kinetic analysis of LapB and the PA domain deletion mutant indicate that the PA domain inhibited enzyme activity of the peptidase domain. Interestingly, the activity of LapB was largely increased by various organic solvents such as ethanol, propanol, and methanol at the concentration of 60% (v/v). CD analysis provided evidence that organic solvents induce the PA domain conformational changes that eliminate the inhibition role. The unique properties indicate the application potential of LapB in the food processing industry.