Syntaxin binding mechanism and disease-causing mutations in Munc18-2

Abstract

Significance Understanding the molecular mechanisms that control secretion from cytotoxic T lymphocytes (CTL) and natural killer (NK) cells is the key for understanding how these cells destroy virally infected and tumourigenic cells. Precisely how mutations in Munc18-2 and syntaxin 11 (Stx11) give rise to loss of CTL and NK function and severe immunodeficiency is poorly understood. In this study we present a crystal structure of human Munc18-2 and analyze the disease-causing mutations. Our findings reveal a mechanism that allows Munc18-2 to selectively bind Stx11 and identify potential surrogate binding partners, which could restore Munc18-Stx function upon IL-2 activation.

SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

“…2A). Consistently, addition of Munc18-2 E132A -a Munc18-2 disease-associated mutant also located within the N lobe of Munc18-2 that reduced binding to STX11 (34,35)-had no effect on the extent of lipid mixing or complete fusion mediated by either STX11-GPI or STX11 H3 -GPI ( Fig. 2A).…”

“…Previous studies have suggested that Munc18-2 can interact with the monomeric Syntaxins STX3 (39,40), STX2 (41), and STX11 (13,(33)(34)(35). However, other SM proteins have distinct binding modes in which they engage either monomeric syntaxins or SNARE complexes (42-45).…”

“…Cells were fixed, permeabilized, and stained using first goat antiMunc18-2 and followed by anti-perforin-Alexa 647 antibodies. binding studies that show that the mutation E132A, which lies within Munc18-2 N-lobe domain, severely affects the interaction with STX11 N-terminal domain (34,35). On the other hand, Munc18-2 R65Q mutant, which binds SNARE complexes more efficiently than Munc18-2 WT but acts in dominant-negative fashion most likely arresting SNAREpin formation (33), had an inhibitory effect on the extent of both lipid mixing and complete fusion mediated by either STX11-GPI or STX11 H3 -GPI.…”

Chaperoning of closed syntaxin-3 through Lys46 and Glu59 in domain 1 of Munc18 proteins is indispensable for mast cell exocytosis

“…Munc18-1 and Munc18-2 are known to bind to syntaxin-2 and syntaxin-3 in addition to syntaxin-11 and neuronal syntaxin-1 (Riento et al, 1998;Tamori et al, 1998). Recent work suggested that syntaxin-3 potentially substitutes for the function of syntaxin-11 in syntaxin-11-deficient mast cells (Hackmann et al, 2013). Thus, we examined a possible change of syntaxin-3 levels in our Munc18-1/2 DKD mast cells, by using a recently generated rabbit monoclonal anti-syntaxin-3 antibody (EPR8543, Abcam).…”

Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

“…However, a recent NMR study on full length Sx1a, including its TMD, reported a higher proportion of the open Sx1a conformation than was observed using the soluble cytosolic domain of Sx1a (Dawidowski & Cafiso, 2013 Although its precise role remains controversial, the importance of SM proteins in disease has been well documented. For example, mutations in Munc18a have been linked with early infantile epileptic encephalopathy (Saitsu et al, 2008(Saitsu et al, , 2010 and Munc18b mutations can result in familial hemophagocytic lymphohistiocytosis type 5 (Cô te et al, 2009;Hackmann et al, 2013). This link between SM proteins and disease has also been confirmed by mutagenesis studies in mice, where deletion of Munc18-1 in mice leads to a complete loss of neurotransmitter secretion from synaptic vesicles throughout development (Verhage et al, 2000).…”