Conformational dynamics of free and membrane-bound human Hsp70 in model cytosolic and endo-lysosomal environments

AbstractThe binding of the major stress-inducible human 70-kDa heat shock protein (Hsp70) to the anionic phospholipid bis-(monoacylglycero)-phosphate (BMP) in the lysosomal membrane is crucial for its impact on cellular pathology in lysosomal storage disorders. However, the conformational features of this protein-lipid complex remain unclear. Here, we apply hydrogen–deuterium exchange mass spectrometry (HDX-MS) to describe the dynamics of the full-length Hsp70 in the cytosol and its conformational changes upon translocation into lysosomes. Using wild-type and W90F mutant proteins, we also map and discriminate the interaction of Hsp70 with BMP and other lipid components of the lysosomal membrane. We identify the N-terminal of the nucleotide binding domain (residues 87–118) as the primary orchestrator of BMP interaction. We show that the conformation of this domain is significantly reorganized in the W90F mutant, explaining its inability to stabilize lysosomal membranes. Overall, our results reveal important new molecular details of the protective effect of Hsp70 in lysosomal storage diseases, which, in turn, could guide future drug development.

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Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis

PeerJ ◽

10.7717/peerj.9408 ◽

2020 ◽

Vol 8 ◽

pp. e9408

Author(s):

Shanshan Wu ◽

Tam T.T.N. Nguyen ◽

Olga V. Moroz ◽

Johan P. Turkenburg ◽

Jens E. Nielsen ◽

...

Keyword(s):

Conformational Changes ◽

Solvent Accessibility ◽

Conformational Dynamics ◽

Structural Heterogeneity ◽

Catalytic Process ◽

Conformational Heterogeneity ◽

X Ray ◽

Exchange Mass Spectrometry ◽

Bacillus Lentus ◽

Hdx Ms

Background Several examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown. Methods Here we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals. Results The NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.

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

10.1101/595553 ◽

2019 ◽

Author(s):

Zainab Ahdash ◽

Euan Pyle ◽

William J. Allen ◽

Robin A. Corey ◽

Ian Collinson ◽

...

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Protein Transport ◽

Atp Hydrolysis ◽

Protein Translocation ◽

Conformational Dynamics ◽

Brownian Ratchet ◽

Exchange Mass Spectrometry ◽

Dependent Transport ◽

Hdx Ms

AbstractThe bacterial Sec translocon is a multi-component 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 unfolded pre-proteins across the membrane. Based on the structure of the machinery, combined with ensemble and single molecule analysis, a diffusional based Brownian ratchet mechanism for protein secretion has been proposed [Allen et al. eLife 2016;5:e15598]. However, the conformational dynamics required to facilitate this mechanism have not yet been fully resolved. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, also ATP-dependent, constriction of SecA around the mature regions of the pre-protein. Thus, ATP binding causes SecY to open and SecA to close, while ATP hydrolysis has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery, and possibly in translocation systems elsewhere. The results highlight the power of HDX-MS for interrogating the dynamic mechanisms of diverse membrane proteins; including their interactions with small molecules such as nucleotides (ATPases and GTPases) and inhibitors (e.g. antibiotics).

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Allosteric regulation of lysosomal enzyme recognition by the cation-independent mannose 6-phosphate receptor

Communications Biology ◽

10.1038/s42003-020-01211-w ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 1

Author(s):

Linda J. Olson ◽

Sandeep K. Misra ◽

Mayumi Ishihara ◽

Kevin P. Battaile ◽

Oliver C. Grant ◽

...

Keyword(s):

Conformational Changes ◽

Protein Trafficking ◽

Lysosomal Enzymes ◽

Lysosomal Storage Diseases ◽

Carbohydrate Binding ◽

Lysosomal Storage ◽

Recombinant Enzymes ◽

X Ray Crystallography ◽

Extracellular Region ◽

Mannose 6 Phosphate

AbstractThe cation-independent mannose 6-phosphate receptor (CI-MPR, IGF2 receptor or CD222), is a multifunctional glycoprotein required for normal development. Through the receptor’s ability to bind unrelated extracellular and intracellular ligands, it participates in numerous functions including protein trafficking, lysosomal biogenesis, and regulation of cell growth. Clinically, endogenous CI-MPR delivers infused recombinant enzymes to lysosomes in the treatment of lysosomal storage diseases. Although four of the 15 domains comprising CI-MPR’s extracellular region bind phosphorylated glycans on lysosomal enzymes, knowledge of how CI-MPR interacts with ~60 different lysosomal enzymes is limited. Here, we show by electron microscopy and hydroxyl radical protein footprinting that the N-terminal region of CI-MPR undergoes dynamic conformational changes as a consequence of ligand binding and different pH conditions. These data, coupled with X-ray crystallography, surface plasmon resonance and molecular modeling, allow us to propose a model explaining how high-affinity carbohydrate binding is achieved through allosteric domain cooperativity.

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Structure and dynamics of the CGRP receptor in apo and peptide-bound forms

Science ◽

10.1126/science.abf7258 ◽

2021 ◽

Vol 372 (6538) ◽

pp. eabf7258 ◽

Cited By ~ 1

Author(s):

Tracy M. Josephs ◽

Matthew J. Belousoff ◽

Yi-Lynn Liang ◽

Sarah J. Piper ◽

Jianjun Cao ◽

...

Keyword(s):

G Protein ◽

Conformational Changes ◽

Conformational Dynamics ◽

Three Dimensional ◽

Cgrp Receptor ◽

Exchange Mass Spectrometry ◽

Gpcr Activation ◽

Calcitonin Gene ◽

Hydrogen Deuterium ◽

Protein Recruitment

G protein–coupled receptors (GPCRs) are key regulators of information transmission between cells and organs. Despite this, we have only a limited understanding of the behavior of GPCRs in the apo state and the conformational changes upon agonist binding that lead to G protein recruitment and activation. We expressed and purified unmodified apo and peptide-bound calcitonin gene–related peptide (CGRP) receptors from insect cells to determine their cryo–electron microscopy (cryo-EM) structures, and we complemented these with analysis of protein conformational dynamics using hydrogen-deuterium exchange mass spectrometry and three-dimensional variance analysis of the cryo-EM data. Together with our previously published structure of the active, Gs-bound CGRP receptor complex, our work provides insight into the mechanisms of class B1 GPCR activation.

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Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

Nature Communications ◽

10.1038/s41467-020-20032-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Ruyu Jia ◽

Chloe Martens ◽

Mrinal Shekhar ◽

Shashank Pant ◽

Grant A. Pellowe ◽

...

Keyword(s):

Conformational Changes ◽

Conformational Transition ◽

Substrate Binding ◽

Conformational Dynamics ◽

Md Simulations ◽

Allosteric Coupling ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Hdx Ms

AbstractProton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.

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Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

10.1101/2020.04.03.023564 ◽

2020 ◽

Author(s):

Ruyu Jia ◽

Chloe Martens ◽

Mrinal Shekhar ◽

Shashank Pant ◽

Grant A. Pellowe ◽

...

Keyword(s):

Conformational Changes ◽

Conformational Transition ◽

Substrate Binding ◽

Conformational Dynamics ◽

Md Simulations ◽

Allosteric Coupling ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Deuterium Exchange Mass Spectrometry ◽

Hdx Ms

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Electron Microscopy in the diagnosis of lysosomal storage diseases

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156316 ◽

1989 ◽

Vol 47 ◽

pp. 866-867

Author(s):

Carole Vogler ◽

Harvey S. Rosenberg

Keyword(s):

Electron Microscopy ◽

Lysosomal Enzyme ◽

Rectal Mucosa ◽

Lysosomal Storage Diseases ◽

Cell Types ◽

Lysosomal Storage ◽

Storage Material ◽

Ultrastructural Examination ◽

Blood Leukocytes ◽

Storage Diseases

Diagnostic procedures for evaluation of patients with lysosomal storage diseases (LSD) seek to identify a deficiency of a responsible lysosomal enzyme or accumulation of a substance that requires the missing enzyme for degradation. Most patients with LSD have progressive neurological degeneration and may have a variety of musculoskeletal and visceral abnormalities. In the LSD, the abnormally diminished lysosomal enzyme results in accumulation of unmetabolized catabolites in distended lysosomes. Because of the subcellular morphology and size of lysosomes, electron microscopy is an ideal tool to study tissue from patients with suspected LSD. In patients with LSD all cells lack the specific lysosomal enzyme but the distribution of storage material is dependent on the extent of catabolism of the substrate in each cell type under normal circumstances. Lysosmal storages diseases affect many cell types and tissues. Storage material though does not accumulate in all tissues and cell types and may be different biochemically and morphologically in different tissues.Conjunctiva, skin, rectal mucosa and peripheral blood leukocytes may show ultrastructural evidence of lysosomal storage even in the absence of clinical findings and thus any of these tissues can be used for ultrastructural examination in the diagnostic evaluation of patients with suspected LSD. Biopsy of skin and conjunctiva are easily obtained and provide multiple cell types including endothelium, epithelium, fibroblasts and nerves for ultrastructural study. Fibroblasts from skin and conjunctiva can also be utilized for the initiation of tissue cultures for chemical assays. Brain biopsy has been largely replaced by biopsy of more readily obtained tissue and by biochemical assays. Such assays though may give equivical or nondiagnostic results and in some lysosomal storage diseases an enzyme defect has not yet been identified and diagnoses can be made only by ultrastructural examination.

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Substrate reduction therapy for lysosomal storage diseases

Acta Paediatrica ◽

10.1080/08035320510028157 ◽

2005 ◽

Vol 94 (0) ◽

pp. 69-75 ◽

Cited By ~ 34

Author(s):

T Cox

Keyword(s):

Lysosomal Storage Diseases ◽

Substrate Reduction Therapy ◽

Lysosomal Storage ◽

Storage Diseases ◽

Substrate Reduction

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Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

10.26434/chemrxiv.5496907 ◽

2017 ◽

Author(s):

Jana Shen ◽

Zhi Yue ◽

Helen Zgurskaya ◽

Wei Chen

Keyword(s):

Molecular Dynamics ◽

Conformational Changes ◽

Transmembrane Domain ◽

Conformational Transition ◽

Conformational Dynamics ◽

Proton Release ◽

Intrinsic Resistance ◽

Constant Ph ◽

Wide Range ◽

Constant Ph Molecular Dynamics

AcrB is the inner-membrane transporter of E. coli AcrAB-TolC tripartite efflux complex, which plays a major role in the intrinsic resistance to clinically important antibiotics. AcrB pumps a wide range of toxic substrates by utilizing the proton gradient between periplasm and cytoplasm. Crystal structures of AcrB revealed three distinct conformational states of the transport cycle, substrate access, binding and extrusion, or loose (L), tight (T) and open (O) states. However, the specific residue(s) responsible for proton binding/release and the mechanism of proton-coupled conformational cycling remain controversial. Here we use the newly developed membrane hybrid-solvent continuous constant pH molecular dynamics technique to explore the protonation states and conformational dynamics of the transmembrane domain of AcrB. Simulations show that both Asp407 and Asp408 are deprotonated in the L/T states, while only Asp408 is protonated in the O state. Remarkably, release of a proton from Asp408 in the O state results in large conformational changes, such as the lateral and vertical movement of transmembrane helices as well as the salt-bridge formation between Asp408 and Lys940 and other sidechain rearrangements among essential residues.Consistent with the crystallographic differences between the O and L protomers, simulations offer dynamic details of how proton release drives the O-to-L transition in AcrB and address the controversy regarding the proton/drug stoichiometry. This work offers a significant step towards characterizing the complete cycle of proton-coupled drug transport in AcrB and further validates the membrane hybrid-solvent CpHMD technique for studies of proton-coupled transmembrane proteins which are currently poorly understood.

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Molecular Basis of Glucose Transport Mechanism in Plants

10.26434/chemrxiv.8049848.v1 ◽

2019 ◽

Cited By ~ 2

Author(s):

Balaji Selvam ◽

Ya-Chi Yu ◽

Liqing Chen ◽

Diwakar Shukla

Keyword(s):

Molecular Dynamics ◽

Free Energy ◽

Molecular Dynamics Simulations ◽

Conformational Changes ◽

Transport Mechanism ◽

Conformational Dynamics ◽

Site Directed Mutagenesis ◽

Free Energy Barrier ◽

Transport Cycle ◽

Dynamics Simulations

The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.

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