Structural insight into the calcium ion modulated interdomain electron transfer in cellobiose dehydrogenase

AbstractCellobiose dehydrogenase (CDH) from wood degrading fungi represents a subclass of oxidoreductases with unique properties. Consisting of two domains exhibiting interdomain electron transfer, this is the only known flavocytochrome involved in wood degradation. High resolution structures of the separated domains were solved, but the overall architecture of the intact protein and the exact interface of the two domains is unknown. Recently, it was shown that divalent cations modulate the activity of CDH and its pH optimum and a possible mechanism involving bridging of negative charges by calcium ions was proposed. Here we provide a structural explanation of this phenomenon confirming the interaction between negatively charged surface patches and calcium ions at the domain interface

Structural Characterization of Phosducin and Its Complex with the 14-3-3 Protein

Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in a phosphorylation-dependent manner, including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, time-resolved fluorescence spectroscopy, and hydrogen-deuterium exchange coupled to mass spectrometry. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3-binding motifs are located, is an intrinsically disordered protein that reduces its flexibility in several regions without undergoing dramatic disorder-to-order transition upon binding to 14-3-3. Our data also indicate that the C-terminal domain of Pdc interacts with the outside surface of the 14-3-3 dimer through the region involved in G$_t$βγ binding. In conclusion, we show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-dependent inhibition of Pdc function

Interdomain electron transfer in cellobiose dehydrogenase is governed by surface electrostatics

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In-solution structure and oligomerization of human histone deacetylase 6 – an integrative approach

Human histone deacetylase 6 (HDAC6) is a structurally unique, multido-main protein implicated in a variety of physiological processes includingcytoskeletal remodelling and the maintenance of cellular homeostasis. Ourcurrent understanding of the HDAC6 structure is limited to isolateddomains, and a holistic picture of the full-length protein structure, includ-ing possible domain interactions, is missing. Here, we used an integrativestructural biology approach to build a solution model of HDAC6 by com-bining experimental data from several orthogonal biophysical techniquescomplemented by molecular modelling. We show that HDAC6 is bestdescribed as a mosaic of folded and intrinsically disordered domains thatin-solution adopts an ensemble of conformations without any stable inter-actions between structured domains. Furthermore, HDAC6 forms dimers/higher oligomers in a concentration-dependent manner, and its oligomer-ization is mediated via the positively charged N-terminal microtubule-binding domain. Our findings provide the first insights into the structure offull-length human HDAC6 and can be used as a basis for further researchinto structure function and physiological studies of this unique deacetylase

Aspartic Protease Nepenthesin‑1 as a Tool for Digestion in Hydrogen/Deuterium Exchange Mass Spectrometry

Hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) utilizes enzymatic digestion of proteins to localize the information about altered exchange patterns in protein structure. The ability of the protease to produce small peptides and overlapping fragments and provide sufficient coverage of the protein sequence is essential for localizing regions of interest. Recently, it was shown that there is an interesting group of proteolytic enzymes from carnivorous pitcher plants of the genus <i>Nepenthes</i>. In this report, we describe successful immobilization and the use of one of these enzymes, nepenthesin-1, in HXMS workflow. In contrast to pepsin, it has different cleavage specificities, and despite its high inherent susceptibility to reducing and denaturing agents, it is very stable upon immobilization and withstands even high concentration of guanidine hydrochloride and reducing agents. We show that denaturing agents can alter digestion by reducing protease activity and/or substrate solubility, and additionally, they influence the trapping of proteolytic peptides onto the reversed phase resin

Human Stress-inducible Hsp70 Has a High Propensity to Form ATP-dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding

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Caldendrin and myosin V regulate synaptic spine apparatus localization via ER stabilization in dendritic spines

Excitatory synapses of principal hippocampal neurons are frequently located on dendritic spines. The dynamic strengthening or weakening of individual inputs results in structural and molecular diversity of dendritic spines. Active spines with large calcium ion (Ca2+) transients are frequently invaded by a single protrusion from the endoplasmic reticulum (ER), which is dynamically transported into spines via the actin-based motor myosin V. An increase in synaptic strength correlates with stable anchoring of the ER, followed by the formation of an organelle referred to as the spine apparatus. Here, we show that myosin V binds the Ca2+ sensor caldendrin, a brain-specific homolog of the well-known myosin V interactor calmodulin. While calmodulin is an essential activator of myosin V motor function, we found that caldendrin acts as an inhibitor of processive myosin V movement. In mouse and rat hippocampal neurons, caldendrin regulates spine apparatus localization to a subset of dendritic spines through a myosin V-dependent pathway. We propose that caldendrin transforms myosin into a stationary F-actin tether that enables the localization of ER tubules and formation of the spine apparatus in dendritic spines

Caldendrin and myosin V regulate synaptic spine apparatus localization via ER stabilization in dendritic spines

Excitatory synapses of principal hippocampal neurons are frequently located on dendritic spines. The dynamic strengthening or weakening of individual inputs results in structural and molecular diversity of dendritic spines. Active spines with large calcium ion (Ca$^{2+}$) transients are frequently invaded by a single protrusion from the endoplasmic reticulum (ER), which is dynamically transported into spines via the actin-based motor myosin V. An increase in synaptic strength correlates with stable anchoring of the ER, followed by the formation of an organelle referred to as the spine apparatus. Here, we show that myosin V binds the Ca$^{2+}$ sensor caldendrin, a brain-specific homolog of the well-known myosin V interactor calmodulin. While calmodulin is an essential activator of myosin V motor function, we found that caldendrin acts as an inhibitor of processive myosin V movement. In mouse and rat hippocampal neurons, caldendrin regulates spine apparatus localization to a subset of dendritic spines through a myosin V-dependent pathway. We propose that caldendrin transforms myosin into a stationary F-actin tether that enables the localization of ER tubules and formation of the spine apparatus in dendritic spines

Coherent diffractive imaging of proteins and viral capsids: simulating MS SPIDOC

MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low- resolution diffractive imaging data (q < 0.3 nm$^{−1}$) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery