Full-length in meso structure and mechanism of rat kynurenine 3-monooxygenase inhibition

AbstractThe structural mechanisms of single-pass transmembrane enzymes remain elusive. Kynurenine 3-monooxygenase (KMO) is a mitochondrial protein involved in the eukaryotic tryptophan catabolic pathway and is linked to various diseases. Here, we report the mammalian full-length structure of KMO in its membrane-embedded form, complexed with compound 3 (identified internally) and compound 4 (identified via DNA-encoded chemical library screening) at 3.0 Å resolution. Despite predictions suggesting that KMO has two transmembrane domains, we show that KMO is actually a single-pass transmembrane protein, with the other transmembrane domain lying laterally along the membrane, where it forms part of the ligand-binding pocket. Further exploration of compound 3 led to identification of the brain-penetrant compound, 5. We show that KMO is dimeric, and that mutations at the dimeric interface abolish its activity. These results will provide insight for the drug discovery of additional blood-brain-barrier molecules, and help illuminate the complex biology behind single-pass transmembrane enzymes.

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Characterization of nuclear pore complex targeting domains in Pom152 in Saccharomyces cerevisiae

10.1101/2020.06.18.157685 ◽

2020 ◽

Author(s):

Jacqueline T. Brown ◽

Alexandra J. Haraczy ◽

Christopher M. Wilhelm ◽

Kenneth D. Belanger

Keyword(s):

Amino Acids ◽

Nuclear Pore Complex ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Full Length ◽

Nuclear Pore ◽

Amino Terminal ◽

Cytosolic Side ◽

Carboxy Terminal ◽

In Cells

AbstractPom152 is a transmembrane protein within the nuclear pore complex (NPC) of fungi that is important for NPC assembly and structure. Pom152 is comprised of a short amino-terminal region that remains on the cytosolic side of the nuclear envelope (NE) and interacts with NPC proteins, a transmembrane domain, and a large, glycosylated carboxy-terminal domain within the NE lumen that self-assembles to form the NPC membrane ring. Here we show that the N-terminal 200 amino acids of Pom152 that include only the amino-terminal and transmembrane regions of the protein are sufficient for localization to the NPC. Full-length, glycosylation-deficient, and truncated Pom152-GFP chimeras expressed in cells containing endogenous Pom152 localize to both NPCs and cortical endoplasmic reticulum (ER). Expression of Pom152-GFP fusions in cells lacking endogenous Pom152 results in detectable localization at only the NE by full-length and amino-terminal Pom152-GFP fusions, but continued retention at both the NE and ER for a chimera lacking just the carboxy-terminal 377 amino acids. Targeted mutations in the amino-terminal and transmembrane domains did not alter Pom152 localization and neither deletion of Pom152 nor its carboxy-terminal glycosylation sites altered the nuclear protein export rate of an Msn5/Kap142 protein cargo. These data narrow the Pom152 region sufficient for NPC localization and provide evidence that alterations in other domains may impact Pom152 targeting or affinity for the NPC.

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Characterization of nuclear pore complex targeting domains in Pom152 in Saccharomyces cerevisiae

Biology Open ◽

10.1242/bio.057661 ◽

2021 ◽

Author(s):

Jacqueline T. Brown ◽

Alexandra J. Haraczy ◽

Christopher M. Wilhelm ◽

Kenneth D. Belanger

Keyword(s):

Amino Acids ◽

Nuclear Pore Complex ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Full Length ◽

Nuclear Pore ◽

Amino Terminal ◽

Glycosylation Sites ◽

Cytosolic Side ◽

Carboxy Terminal

Pom152 is a transmembrane protein within the nuclear pore complex (NPC) of fungi that is important for NPC assembly and structure. Pom152 is comprised of a short amino-terminal region that remains on the cytosolic side of the nuclear envelope (NE) and interacts with NPC proteins, a transmembrane domain, and a large, glycosylated carboxy-terminal domain within the NE lumen. Here we show that the N-terminal 200 amino acids of Pom152 that include only the amino-terminal and transmembrane regions are sufficient for localization to the NPC. Full-length, glycosylation-deficient, and truncated Pom152-GFP chimeras expressed in cells containing endogenous Pom152 localize to both NPCs and cortical endoplasmic reticulum (ER). Expression of Pom152-GFP fusions in pom152Δ cells results in detectable localization at only the NE by full-length and amino-terminal Pom152-GFP fusions, but continued retention at both the NE and ER for a chimera lacking just the carboxy-terminal 377 amino acids. Neither deletion of Pom152 nor its carboxy-terminal glycosylation sites altered the nuclear protein export rate of an Msn5/Kap142 protein cargo. These data narrow the Pom152 region sufficient for NPC localization and provide evidence that alterations in other domains may impact Pom152 targeting or affinity for the NPC.

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Cryo-EM structure of human Wntless in complex with Wnt3a

Nature Communications ◽

10.1038/s41467-021-24731-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Qing Zhong ◽

Yanyu Zhao ◽

Fangfei Ye ◽

Zaiyu Xiao ◽

Gaoxingyu Huang ◽

...

Keyword(s):

Transmembrane Domain ◽

Transmembrane Protein ◽

Multiple Interfaces ◽

Wnt Proteins ◽

Binding Partners ◽

Multiple Binding Sites ◽

Evolutionarily Conserved ◽

Multiple Binding ◽

Hydrophobic Tunnel ◽

Conserved Core

AbstractWntless (WLS), an evolutionarily conserved multi-pass transmembrane protein, is essential for secretion of Wnt proteins. Wnt-triggered signaling pathways control many crucial life events, whereas aberrant Wnt signaling is tightly associated with many human diseases including cancers. Here, we report the cryo-EM structure of human WLS in complex with Wnt3a, the most widely studied Wnt, at 2.2 Å resolution. The transmembrane domain of WLS bears a GPCR fold, with a conserved core cavity and a lateral opening. Wnt3a interacts with WLS at multiple interfaces, with the lipid moiety on Wnt3a traversing a hydrophobic tunnel of WLS transmembrane domain and inserting into membrane. A β-hairpin of Wnt3a containing the conserved palmitoleoylation site interacts with WLS extensively, which is crucial for WLS-mediated Wnt secretion. The flexibility of the Wnt3a loop/hairpin regions involved in the multiple binding sites indicates induced fit might happen when Wnts are bound to different binding partners. Our findings provide important insights into the molecular mechanism of Wnt palmitoleoylation, secretion and signaling.

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Cryo-EM structure of the human histamine H1 receptor/Gq complex

Nature Communications ◽

10.1038/s41467-021-22427-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ruixue Xia ◽

Na Wang ◽

Zhenmei Xu ◽

Yang Lu ◽

Jing Song ◽

...

Keyword(s):

Transmembrane Domain ◽

Binding Pocket ◽

Receptor Activation ◽

Intracellular Loop ◽

Cryo Electron Microscopy ◽

Allergy Treatment ◽

Domain 3 ◽

Extracellular Side ◽

Key Residues ◽

Loop 2

AbstractHistamine receptors play important roles in various pathophysiological conditions and are effective targets for anti-allergy treatment, however the mechanism of receptor activation remain elusive. Here, we present the cryo-electron microscopy (cryo-EM) structure of the human H1R in complex with a Gq protein in an active conformation via a NanoBiT tethering strategy. The structure reveals that histamine activates receptor via interacting with the key residues of both transmembrane domain 3 (TM3) and TM6 to squash the binding pocket on the extracellular side and to open the cavity on the intracellular side for Gq engagement in a model of “squash to activate and expand to deactivate”. The structure also reveals features for Gq coupling, including the interaction between intracellular loop 2 (ICL2) and the αN-β junction of Gq/11 protein. The detailed analysis of our structure will provide a framework for understanding G-protein coupling selectivity and clues for designing novel antihistamines.

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Vaccinia Virus A26 and A27 Proteins Form a Stable Complex Tethered to Mature Virions by Association with the A17 Transmembrane Protein

Journal of Virology ◽

10.1128/jvi.01524-08 ◽

2008 ◽

Vol 82 (24) ◽

pp. 12384-12391 ◽

Cited By ~ 30

Author(s):

Amanda R. Howard ◽

Tatiana G. Senkevich ◽

Bernard Moss

Keyword(s):

Vaccinia Virus ◽

Matrix Protein ◽

Transmembrane Domain ◽

Noncovalent Complex ◽

Transmembrane Protein ◽

Amino Acid Identity ◽

Noncovalent Interaction ◽

Terminal Segment ◽

Stable Complex ◽

Cowpox Virus

ABSTRACT During vaccinia virus replication, mature virions (MVs) are wrapped with cellular membranes, transported to the periphery, and exported as extracellular virions (EVs) that mediate spread. The A26 protein is unusual in that it is present in MVs but not EVs. This distribution led to a proposal that A26 negatively regulates wrapping. A26 also has roles in the attachment of MVs to the cell surface and incorporation of MVs into proteinaceous A-type inclusions in some orthopoxvirus species. However, A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV, regulates incorporation of the MV into inclusions, and possibly prevents EV formation. Here, we provide evidence that A26 forms a disulfide-bonded complex with A27 that is anchored to the MV through a noncovalent interaction with the A17 transmembrane protein. In the absence of A27, A26 was unstable, and only small amounts were detected. The interaction of A26 with A27 depended on a C-terminal segment of A26 with 45% amino acid identity to A27. Deletion of A26 failed to enhance EV formation by vaccinia virus, as had been predicted. Nevertheless, the interaction of A26 and A27 may have functional significance, since each is thought to mediate binding to cells through interaction with laminin and heparan sulfate, respectively. We also found that A26 formed a noncovalent complex with A25, a truncated form of the cowpox virus A-type inclusion matrix protein. The latter association suggests a mechanism for incorporation of virions into A-type inclusions in other orthopoxvirus strains.

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The costimulatory activity of Tim-3 requires Akt and MAPK signaling and its recruitment to the immune synapse

Science Signaling ◽

10.1126/scisignal.aba0717 ◽

2021 ◽

Vol 14 (687) ◽

pp. eaba0717

Author(s):

Shunsuke Kataoka ◽

Priyanka Manandhar ◽

Judong Lee ◽

Creg J. Workman ◽

Hridesh Banerjee ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Cell Receptor ◽

Mapk Signaling ◽

Inhibitory Protein ◽

Immune Synapse

Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic activation, including during chronic infection and in solid tumors. Thus, Tim-3 is generally thought of as an inhibitory protein. We and others previously reported that under some circumstances, Tim-3 exerts paradoxical costimulatory activity in T cells (and other cells), including enhancement of the phosphorylation of ribosomal S6 protein. Here, we examined the upstream signaling pathways that control Tim-3–mediated increases in phosphorylated S6 in T cells. We also defined the localization of Tim-3 relative to the T cell immune synapse and its effects on downstream signaling. Recruitment of Tim-3 to the immune synapse was mediated exclusively by the transmembrane domain, replacement of which impaired the ability of Tim-3 to costimulate T cell receptor (TCR)–dependent S6 phosphorylation. Furthermore, enforced localization of the Tim-3 cytoplasmic domain to the immune synapse in a chimeric antigen receptor still enabled T cell activation. Together, our findings are consistent with a model whereby Tim-3 enhances TCR-proximal signaling under acute conditions.

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Canine Influenza Virus is Mildly Restricted by Canine Tetherin Protein

Viruses ◽

10.3390/v10100565 ◽

2018 ◽

Vol 10 (10) ◽

pp. 565

Author(s):

Yun Zheng ◽

Xiangqi Hao ◽

Qingxu Zheng ◽

Xi Lin ◽

Xin Zhang ◽

...

Keyword(s):

Influenza Virus ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Cellular Damage ◽

Interferon Response ◽

Type I ◽

Canine Influenza Virus ◽

Immune Factor ◽

Canine Influenza ◽

The Impact

Tetherin (BST2/CD317/HM1.24) has emerged as a key host-cell ·defence molecule that acts by inhibiting the release and spread of diverse enveloped virions from infected cells. We analysed the biological features of canine tetherin and found it to be an unstable hydrophilic type I transmembrane protein with one transmembrane domain, no signal peptide, and multiple glycosylation and phosphorylation sites. Furthermore, the tissue expression profile of canine tetherin revealed that it was particularly abundant in immune organs. The canine tetherin gene contains an interferon response element sequence that can be regulated and expressed by canine IFN-α. A CCK-8 assay showed that canine tetherin was effective in helping mitigate cellular damage caused by canine influenza virus (CIV) infection. Additionally, we found that the overexpression of canine tetherin inhibited replication of the CIV and that interference with the canine tetherin gene enhanced CIV replication in cells. The impact of canine tetherin on CIV replication was mild. However, these results elucidate the role of the innate immune factor, canine tetherin, during CIV infection for the first time.

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Two transmembrane dimers of the bovine papillomavirus E5 oncoprotein clamp the PDGF β receptor in an active dimeric conformation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1705622114 ◽

2017 ◽

Vol 114 (35) ◽

pp. E7262-E7271 ◽

Cited By ~ 5

Author(s):

Alexander G. Karabadzhak ◽

Lisa M. Petti ◽

Francisco N. Barrera ◽

Anne P. B. Edwards ◽

Andrés Moya-Rodríguez ◽

...

Keyword(s):

Transmembrane Domain ◽

Transmembrane Protein ◽

Receptor Activation ◽

Bovine Papillomavirus ◽

Detailed Model ◽

E5 Protein ◽

Membrane Modeling ◽

E5 Oncoprotein ◽

Β Receptor ◽

Dynamics Simulations

The dimeric 44-residue E5 protein of bovine papillomavirus is the smallest known naturally occurring oncoprotein. This transmembrane protein binds to the transmembrane domain (TMD) of the platelet-derived growth factor β receptor (PDGFβR), causing dimerization and activation of the receptor. Here, we use Rosetta membrane modeling and all-atom molecular dynamics simulations in a membrane environment to develop a chemically detailed model of the E5 protein/PDGFβR complex. In this model, an active dimer of the PDGFβR TMD is sandwiched between two dimers of the E5 protein. Biochemical experiments showed that the major PDGFβR TMD complex in mouse cells contains two E5 dimers and that binding the PDGFβR TMD to the E5 protein is necessary and sufficient to recruit both E5 dimers into the complex. These results demonstrate how E5 binding induces receptor dimerization and define a molecular mechanism of receptor activation based on specific interactions between TMDs.

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Structural basis for cholesterol transport-like activity of the Hedgehog receptor Patched

10.1101/352443 ◽

2018 ◽

Cited By ~ 3

Author(s):

Yunxiao Zhang ◽

David P. Bulkley ◽

Kelsey J. Roberts ◽

Yao Xin ◽

Daniel E. Asarnow ◽

...

Keyword(s):

Plasma Membrane ◽

Basal Cell Carcinoma ◽

Cell Carcinoma ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Extracellular Domain ◽

Structural Basis ◽

Cholesterol Transport ◽

Common Cause ◽

Common Receptor

AbstractHedgehog protein signals mediate tissue patterning and maintenance via binding to and inactivation of their common receptor Patched, a twelve-transmembrane protein that otherwise would suppress activity of the seven-transmembrane protein, Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. The extracellular domain mediates association of Patched monomers in an unusual dimeric architecture that implies curvature in the associated membrane. A central conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Patched expression indeed reduces cholesterol activity in the inner leaflet of the plasma membrane, in a manner antagonized by Hedgehog stimulation and with implications for regulation of Smoothened.

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Progressive axonopathy when oligodendrocytes lack the myelin protein CMTM5

10.1101/2021.11.22.469514 ◽

2021 ◽

Author(s):

Tobias J Buscham ◽

Maria A. Eichel-Vogel ◽

Anna M Steyer ◽

Olaf Jahn ◽

Nicola Strenzke ◽

...

Keyword(s):

Early Onset ◽

Wallerian Degeneration ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Myelin Proteins ◽

Myelin Protein ◽

Impulse Propagation ◽

Functional Relevance

Oligodendrocytes facilitate rapid impulse propagation along the axons they myelinate and support their long-term integrity. However, the functional relevance of many myelin proteins has remained unknown. Here we find that expression of the tetraspan-transmembrane protein CMTM5 (Chemokine-like factor-like MARVEL-transmembrane domain containing protein 5) is highly enriched in oligodendrocytes and CNS myelin. Genetic disruption of the Cmtm5-gene in oligodendrocytes of mice does not impair the development or ultrastructure of CNS myelin. However, oligodendroglial Cmtm5-deficiency causes an early-onset progressive axonopathy, which we also observe in global and in tamoxifen-induced oligodendroglial Cmtm5-mutants. Presence of the Wlds mutation ameliorates the axonopathy, implying a Wallerian degeneration-like pathomechanism. These results indicate that CMTM5 is involved in the function of oligodendrocytes to maintain axonal integrity rather than myelin biogenesis.

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