Structural insights into the activation mechanism of dynamin-like EHD ATPases

Melo, Arthur A.; Hegde, Balachandra G.; Shah, Claudio; Larsson, Elin; Isas, J. Mario; Kunz, Séverine; Lundmark, Richard; Langen, Ralf; Daumke, Oliver

doi:10.1073/pnas.1614075114

Cited by 38 publications

(85 citation statements)

References 36 publications

(45 reference statements)

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“…S7A, Bottom Right). The proposed reorientation is in agreement with the crystal structure of the structurally related N-terminally deleted EHD4 (29). The described conformational change allows the N terminus of EHD2 to be inserted into the lipid layer.…”

Section: Discussionsupporting

confidence: 84%

EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation

Hoernke

Mohan

Larsson

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The EH-domain-containing protein 2 (EHD2) is a dynamin-related ATPase that confines caveolae to the cell surface by restricting the scission and subsequent endocytosis of these membrane pits. For this, EHD2 is thought to first bind to the membrane, then to oligomerize, and finally to detach, in a stringently regulated mechanistic cycle. It is still unclear how ATP is used in this process and whether membrane binding is coupled to conformational changes in the protein. Here, we show that the regulatory N-terminal residues and the EH domain keep the EHD2 dimer in an autoinhibited conformation in solution. By significantly advancing the use of infrared reflection-absorption spectroscopy, we demonstrate that EHD2 adopts an open conformation by tilting the helical domains upon membrane binding. We show that ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is related to detachment of EHD2 from the membrane. Finally, we demonstrate that the regulation of EHD2 oligomerization in a membrane-bound state is crucial to restrict caveolae dynamics in cells.EHD2 | caveolae | membrane-reshaping protein | membrane-bound protein structure | infrared reflection-absorption spectroscopy

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Section: Discussionsupporting

confidence: 84%

EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation

Hoernke

Mohan

Larsson

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Eps15‐homology domain‐containing proteins (EHDs) are often included in the dynamin superfamily owing to their micromolar affinities for nucleotides, self‐assembly, stimulated hydrolysis, and roles in membrane remodeling events . Furthermore, the helical bundle adjacent to the G domain undergoes a significant conformational change, similar to dynamins in general . However, while the EHD catalytic core adopts a G domain fold, these enzymes specifically bind and hydrolyze ATP, rather than GTP, due to a steric occlusion in their nucleotide binding pockets …”

Section: Strays Waifs and Wannabes: An Existential Crisis Of What Dementioning

confidence: 99%

The structural biology of the dynamin‐related proteins: New insights into a diverse, multitalented family

Ford

Chappie

2019

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Dynamin‐related proteins are multidomain, mechanochemical GTPases that self‐assemble and orchestrate a wide array of cellular processes. Over the past decade, structural insights from X‐ray crystallography and cryo‐electron microscopy have reshaped our mechanistic understanding of these proteins. Here, we provide a historical perspective on these advances that highlights the structural attributes of different dynamin family members and explores how these characteristics affect GTP hydrolysis, conformational coupling and oligomerization. We also discuss a number of lingering challenges remaining in the field that suggest future directions of study.

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“…Accessibilities of the spin labels to O 2 and NiEDDA further supported the notion that this region of the helical domain inserts into the membrane [10]. A subsequent study [117] found that this membrane binding region also included additional amino acids in the tip region, as the helical domain can undergo a domain rotation that allows additional amino acids to come into contact with the membrane (Fig. 7).…”

Section: Non-amyloidogenic Proteins and Membrane Remodelingmentioning

confidence: 61%

“…7). Using SDSL and EPR we tested whether this region was responsible for recruiting EHD2 to the membrane [10, 117]. First, the three native cysteines in EHD2 were mutated to serines.…”

Section: Non-amyloidogenic Proteins and Membrane Remodelingmentioning

confidence: 99%

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR

Varkey

Langen

2017

Journal of Magnetic Resonance

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The advancement in site directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single protein can generate different type of membrane curvatures using specific conformations for specific membrane structures and how EPR is a versatile tool well-suited to analyze subtle alterations in structures under such modifying conditions which otherwise would have been difficult using other biophysical tools.

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Structural insights into the activation mechanism of dynamin-like EHD ATPases

Cited by 38 publications

References 36 publications

EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation

EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation

The structural biology of the dynamin‐related proteins: New insights into a diverse, multitalented family

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR

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