Structure of a volume-regulated heteromeric LRRC8A/C channel

Volume-regulated anion channels (VRACs) participate in the cellular response to osmotic swelling. These membrane proteins consist of heteromeric assemblies of LRRC8 subunits, whose compositions determine permeation properties. Although structures of the obligatory LRRC8A, also referred to as SWELL1, have previously defined the architecture of VRACs, the organization of heteromeric channels has remained elusive. Here we have addressed this question by the structural characterization of murine LRRC8A/C channels. Like LRRC8A, these proteins assemble as hexamers. Despite 12 possible arrangements, we find a predominant organization with an A:C ratio of two. In this assembly, four LRRC8A subunits cluster in their preferred conformation observed in homomers, as pairs of closely interacting proteins that stabilize a closed state of the channel. In contrast, the two interacting LRRC8C subunits show a larger flexibility, underlining their role in the destabilization of the tightly packed A subunits, thereby enhancing the activation properties of the protein

Structures of atypical chemokine receptor 3 reveal the basis for its promiscuity and signaling bias

Both CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are activated by the chemokine CXCL12 yet evoke distinct cellular responses. CXCR4 is a canonical G protein–coupled receptor (GPCR), whereas ACKR3 is intrinsically biased for arrestin. The molecular basis for this difference is not understood. Here, we describe cryo-EM structures of ACKR3 in complex with CXCL12, a more potent CXCL12 variant, and a small-molecule agonist. The bound chemokines adopt an unexpected pose relative to those established for CXCR4 and observed in other receptor-chemokine complexes. Along with functional studies, these structures provide insight into the ligand-binding promiscuity of ACKR3, why it fails to couple to G proteins, and its bias toward β-arrestin. The results lay the groundwork for understanding the physiological interplay of ACKR3 with other GPCRs

ADAM17 Silencing in Mouse Colon Carcinoma Cells: The Effect on Tumoricidal Cytokines and Angiogenesis

ADAM17 (a disintegrin and metalloprotease 17) is a major sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules and is often overexpressed in malignant cells. It is generally accepted that ADAM17 promotes tumor development via activating growth factors from the EGF family, thus facilitating autocrine stimulation of tumor cell proliferation and migration. Here we show, using MC38CEA murine colon carcinoma model, that ADAM17 also regulates tumor angiogenesis and cytokine profile. When ADAM17 was silenced in MC38CEA cells, in vivo tumor growth and in vitro cell motility were significantly diminished, but no effect was seen on in vitro cell proliferation. ADAM17-silencing was accompanied by decreased in vitro expression of vascular endothelial growth factor-A and matrix metalloprotease-9, which was consistent with the limited angiogenesis and slower growth seen in ADAM17-silenced tumors. Among the growth factors susceptible to shedding by ADAM17, neuregulin-1 was the only candidate to mediate the effects of ADAM17 on MC38CEA motility and tumor angiogenesis. Concentrations of TNF and IFN gamma, cytokines that synergistically induced proapoptotic effects on MC38CEA cells, were significantly elevated in the lysates of ADAM17-silenced tumors compared to mock transfected controls, suggesting a possible role for ADAM17 in host immune suppression. These results introduce new, complex roles of ADAM17 in tumor progression, including its impact on the anti-tumor immune response

Zastosowanie konformacyjnie specyficznych fragmentów przeciwciał w badaniach strukturalnych kanału magnezowego CorA

CorA z Thermatoga maritima to pentameryczny kanał jonowy odpowiedzialny za homeostazę jonów magnezu w komórce. Nietypowa budowa kanału, z dużą, N-końcową domeną cytoplazmatyczną oraz domeną C-końcową tworzącą por, jest silnie zakonserwowana wśród prokariotów. Struktura zamkniętego stanu kanału CorA została rozwiązana w 2005 roku, natomiast struktura konformacji otwartej, zdolnej do przewodzenia jonów nadal pozostaje nieznana. Niniejsza praca opisuje próbę rozwiązana struktury otwartego stanu CorA z zastosowaniem konformacyjnie specyficznych, syntetycznych fragmentów przeciwciał jako czaperonów krystalizacji. Fragmenty przeciwciał uzyskano z wykorzystaniem nowatorskiej metody opartej o ekspresję fagową, która wykorzystuje nanodyski jako nośniki białek membranowych.CorA wprowadzono do biotynylowanych nanodysków o średnicy ok. 8 nm. Tak uzyskane struktury białkowo-lipidowe wiążą się na złożu opłaszczonym streptawidyną, a ich elucja możliwa jest z użyciem detergentu. Po 5 rundach selekcji przeanalizowano 96 klonów pod kątem specyficznego wiązania do CorA w nanodyskach. Ich sekwencjonowanie ujawniło obecność 27 unikalnych fragmentów przeciwciał, które następnie zostały wyekspresjonowane i oczyszczone. Wszystkie oczyszczone przeciwciała specyficznie wiążą CorA w detergencie oraz większość z nich wykazuje wiązanie zależne od jonów magnezu, co świadczy o ich specyficzności konformacyjnej. Uzyskane przeciwciała tworzą stabilne kompleksy z CorA, a część również z rozpuszczalną domeną N-terminalną. Ich zastosowanie jako czaperonów krystalizacji pozwala na uzyskanie nowych morfologii kryształów, które po optymalizacji mogą zostać użyte do analizy dyfrakcji promieniowania X.Dzięki ekspresji fagowej uzyskano panel potencjalnych czaperonów krystalizacji, przy czym wiele z nich wiąże się tylko do wybranych konformacji CorA. Zastosowanie przeciwciał usprawnia krystalizację, a uzyskane kryształy mogą potencjalnie dostarczyć informacji strukturalnych o otwartej konformacji kanału CorA.CorA from Thermatoga maritima is a pentameric divalent ion channel responsible for magnesium homeostasis. Its unusual topology, with a bulky and highly charged cytoplasmic domain and pore-forming C-terminus, is well conserved among prokaryotes. While the structure of the closed state of the channel was solved in 2005, the structure of the open, conductive state still remains elusive. This study presents an attempt to resolve the structure of the open state of CorA using conformation-specific, synthetic antibody fragments (sABs) generated using a novel, phage display protocol in NDs as crystallization chaperones.CorA is incorporated into biotinylated, lipid filled nanodiscs that are 8 nm in diameter. It was shown that CorA in NDs is efficiently captured onto and released from the streptavidin-coated solid surface. After five rounds of biopanning, 96 clones were investigated for binding towards CorA with phage ELISA. Sequence analysis of positive clones revealed 27 unique sABs that bind to CorA with high specificity. The unique sABs were then cloned into expression vectors, expressed in E.coli and purified. All purified sABs retained their binding towards CorA in detergent, and the majority of the generated sABs show magnesium-dependent binding, which confirms their conformation-specificity. The generated sABs form stable complexes with full length CorA and CorA N-terminal domain. Application of sABs as crystallization chaperones facilitate formation of crystals of new, unique morphologies.Phage display selection allowed generation of a panel of potential crystallization chaperones, many of which are conformation-specific. The generated sABs were shown to facilitate crystallization of CorA. Acquired crystals can therefore be used for X-ray diffraction analysis and can potentially allow us to resolve the structure of CorA in the open, conductive conformation

Cryo-EM structures of the TTYH family reveal a novel architecture for lipid interactions

The Tweety homologs (TTYHs) are members of a conserved family of eukaryotic membrane proteins that are abundant in the brain. The three human paralogs were assigned to function as anion channels that are either activated by Ca2+ or cell swelling. To uncover their unknown architecture and its relationship to function, we have determined the structures of human TTYH1–3 by cryo-electron microscopy. All structures display equivalent features of a dimeric membrane protein that contains five transmembrane segments and an extended extracellular domain. As none of the proteins shows attributes reminiscent of an anion channel, we revisited functional experiments and did not find any indication of ion conduction. Instead, we find density in an extended hydrophobic pocket contained in the extracellular domain that emerges from the lipid bilayer, which suggests a role of TTYH proteins in the interaction with lipid-like compounds residing in the membrane

Allosteric modulation of LRRC8 channels by targeting their cytoplasmic domains

Members of the LRRC8 family form heteromeric assemblies, which function as volume-regulated anion channels. These modular proteins consist of a transmembrane pore and cytoplasmic leucine-rich repeat (LRR) domains. Despite their known molecular architecture, the mechanism of activation and the role of the LRR domains in this process has remained elusive. Here we address this question by generating synthetic nanobodies, termed sybodies, which target the LRR domain of the obligatory subunit LRRC8A. We use these binders to investigate their interaction with homomeric LRRC8A channels by cryo-electron microscopy and the consequent effect on channel activation by electrophysiology. The five identified sybodies either inhibit or enhance activity by binding to distinct epitopes of the LRR domain, thereby altering channel conformations. In combination, our work provides a set of specific modulators of LRRC8 proteins and reveals the role of their cytoplasmic domains as regulators of channel activity by allosteric mechanisms

Structure of a volume-regulated heteromeric LRRC8A/C channel

Volume-regulated anion channels (VRACs) participate in the cellular response to osmotic swelling. These membrane proteins consist of heteromeric assemblies of LRRC8 subunits, whose compositions determine permeation properties. Although structures of the obligatory LRRC8A, also referred to as SWELL1, have previously defined the architecture of VRACs, the organization of heteromeric channels has remained elusive. Here we have addressed this question by the structural characterization of murine LRRC8A/C channels. Like LRRC8A, these proteins assemble as hexamers. Despite 12 possible arrangements, we find a predominant organization with an A:C ratio of two. In this assembly, four LRRC8A subunits cluster in their preferred conformation observed in homomers, as pairs of closely interacting proteins that stabilize a closed state of the channel. In contrast, the two interacting LRRC8C subunits show a larger flexibility, underlining their role in the destabilization of the tightly packed A subunits, thereby enhancing the activation properties of the protein.ISSN:1545-9993ISSN:1545-998

Cryo-EM structures and functional properties of CALHM channels of the human placenta

The transport of substances across the placenta is essential for the development of the fetus. Here, we were interested in the role of channels of the calcium homeostasis modulator (CALHM) family in the human placenta. By transcript analysis, we found the paralogs CALHM2, 4, and 6 to be highly expressed in this organ and upregulated during trophoblast differentiation. Based on electrophysiology, we observed that activation of these paralogs differs from the voltage- and calcium-gated channel CALHM1. Cryo-EM structures of CALHM4 display decameric and undecameric assemblies with large cylindrical pore, while in CALHM6 a conformational change has converted the pore shape into a conus that narrows at the intracellular side, thus describing distinct functional states of the channel. The pore geometry alters the distribution of lipids, which occupy the cylindrical pore of CALHM4 in a bilayer-like arrangement whereas they have redistributed in the conical pore of CALHM6 with potential functional consequences

Cryo-EM structures and functional properties of CALHM channels of the human placenta.

The transport of substances across the placenta is essential for the development of the fetus. Here, we were interested in the role of channels of the calcium homeostasis modulator (CALHM) family in the human placenta. By transcript analysis, we found the paralogs CALHM2, 4, and 6 to be highly expressed in this organ and upregulated during trophoblast differentiation. Based on electrophysiology, we found that activation of these paralogs differs from the voltage- and calcium-gated channel CALHM1. Cryo-EM structures of CALHM4 display decameric and undecameric assemblies with large cylindrical pore, while in CALHM6 a conformational change has converted the pore shape into a conus that narrows at the intracellular side, thus describing distinct functional states of the channel. The pore geometry alters the distribution of lipids, which occupy the cylindrical pore of CALHM4 in a bilayer-like arrangement whereas they have redistributed in the conical pore of CALHM6 with potential functional consequences