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The activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure.

ABSTRACT: The activating NKG2D receptor plays a critical role in innate and adaptive immune responses by natural killer cells and subpopulations of T cells. The human receptor assembles with the DAP10 signaling dimer, and it is thought that one NKG2D homodimer pairs with a single DAP10 dimer by formation of two salt bridges between charged transmembrane (TM) residues. However, direct stoichiometry measurements demonstrated that one NKG2D homodimer assembles with four DAP10 chains. Selective mutation of one of the basic TM residues of NKG2D resulted in loss of two DAP10 chains, indicating that each TM arginine serves as an interaction site for a DAP10 dimer. Assembly of the hexameric structure was cooperative because this mutation also significantly reduced NKG2D dimerization. A monomeric NKG2D TM peptide was sufficient for assembly with a DAP10 dimer, indicating that the interaction between these proteins occurs in the membrane environment. Formation of a three-helix interface among the TM domains involved ionizable residues from all three chains, the TM arginine of NKG2D and both TM aspartic acids of the DAP10 dimer. The organization of the TM domains thus shows similarities to the T cell antigen receptor-CD3 complex, in particular to the six-chain assembly intermediate between T cell antigen receptor and the CD3delta epsilon and CD3gamma epsilon dimers. Binding of a single ligand can thus result in phosphorylation of four DAP10 chains, which may be relevant for the sensitivity of NKG2D receptor signaling, in particular in situations of low ligand density.

SUBMITTER: Garrity D

PROVIDER: S-EPMC1140444 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

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The activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure.

Garrity David D Call Matthew E ME Feng Jianwen J Wucherpfennig Kai W KW

Proceedings of the National Academy of Sciences of the United States of America 20050513 21

The activating NKG2D receptor plays a critical role in innate and adaptive immune responses by natural killer cells and subpopulations of T cells. The human receptor assembles with the DAP10 signaling dimer, and it is thought that one NKG2D homodimer pairs with a single DAP10 dimer by formation of two salt bridges between charged transmembrane (TM) residues. However, direct stoichiometry measurements demonstrated that one NKG2D homodimer assembles with four DAP10 chains. Selective mutation of on ...[more]

PMID: 15894612

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Project description:The S. cerevisiae plasma membrane H+-ATPase, Pma1, is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma1 (MCP). Like other plasma membrane H+-ATPases, Pma1 assembles and functions as a hexamer, a property unique to this subfamily among the larger family of P-type ATPases. It has been unclear how Pma1 organizes the yeast membrane into MCP microdomains, or why it is that Pma1 needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. Here we report a high-resolution cryo-EM study of native Pma1 hexamers embedded in endogenous lipids. Remarkably, we found that the Pma1 hexamer encircles a liquid-crystalline membrane domain composed of 57 ordered lipid molecules. The Pma1-encircled lipid patch structure likely serves as the building block of the MCP. At pH 7.4, the carboxyl-terminal regulatory α-helix binds to the phosphorylation domains of two neighboring Pma1 subunits, locking the hexamer in the autoinhibited state. The regulatory helix becomes disordered at lower pH, leading to activation of the Pma1 hexamer. The activation process is accompanied by a 6.7 Å downward shift and a 40° rotation of transmembrane helices 1 and 2 that line the proton translocation path. The conformational changes have enabled us to propose a detailed mechanism for ATP-hydrolysis-driven proton pumping across the plasma membrane. Our structures will facilitate the development of antifungal drugs that target this essential protein. The plasma membrane H+ -ATPase is responsible for maintenance of the plasma membrane potential, which provides energy for the transport of nutrients, and the plasma membrane H+ -ATPase in S. cerevisiae (Pma1) is a P3A-type ATPase that assembles and functions as a hexamer. Here, the authors present the cryo-EM structures of autoinhibited and activated native Pma1 hexamers purified with endogenous lipids and they propose a mechanism for proton pumping across the membrane by this family of H+ -ATPases.

Project description:Pseudomonas aeruginosa CtpA is a carboxyl-terminal processing protease that partners with the outer membrane lipoprotein LbcA to degrade at least five cell wall-associated proteins, four of which are cell wall hydrolases. This activity plays an important role in supporting P. aeruginosa virulence in a mouse model of acute pneumonia. However, almost nothing is known about the molecular mechanisms underlying CtpA and LbcA function. Here, we used structural analysis to show that CtpA alone assembles into an inactive hexamer comprising a trimer of dimers, which limits its substrate access and prevents nonspecific degradation. The adaptor protein LbcA is a right-handed open spiral with 11 tetratricopeptide repeats, which might wrap around a substrate to deliver it to CtpA for degradation. By structure-guided mutagenesis and functional assays, we also showed that the interfaces of the CtpA trimer of dimers and an N-terminal helix of LbcA are important for LbcA-mediated substrate degradation by CtpA both in vitro and in vivo. This work improves our understanding of the molecular mechanism of the LbcA-CtpA proteolytic system and reveals some striking differences from the arrangements found in some other bacterial CTPs. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are found in all three domains of life. In bacteria, some CTPs have been associated with virulence, raising the possibility that they could be therapeutic targets. However, relatively little is known about their molecular mechanisms of action. In Pseudomonas aeruginosa, CtpA supports virulence by working in complex with the outer membrane lipoprotein LbcA to degrade cell wall hydrolases. Here, we report structure-function analyses of CtpA and LbcA, which reveals that CtpA assembles into an inactive hexamer comprising a trimer of dimers. LbcA is monomeric, with the first N-terminal helix important for binding to and activating CtpA, followed by a spiral structure composed of 11 tetratricopeptide repeats, which could wrap around a substrate for delivery to CtpA. This work reveals a unique mutimeric arrangement for a CTP and insight into how the important LbcA-CtpA proteolytic system functions.

Dataset Information

The activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure.

Publications

The activating NKG2D receptor assembles in the membrane with two signaling dimers into a hexameric structure.

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