Elucidation of the molecular interactions that enable stable assembly and structural diversity in multicomponent immune receptors

Abstract

Multicomponent immune receptors are essential complexes in which distinct ligand-recognition and signaling subunits are held together by interactions between acidic and basic residues of their transmembrane helices. A 2:1 acidic-to-basic motif in the transmembrane domains of the subunits is necessary and sufficient to assemble these receptor complexes. Here, we study a prototype for these receptors, a DAP12-NKG2C 2:1 heterotrimeric complex, in which the two DAP12 subunits each contribute a single transmembrane Asp residue, and the NKG2C subunit contributes a Lys to form the complex. DAP12 can also associate with 20 other subunits using a similar motif. Here, we use molecular-dynamics simulations to understand the basis for the high affinity and diversity of interactions in this group of receptors. Simulations of the transmembrane helices with differing protonation states of the Asp-Asp-Lys triad identified a structurally stable interaction in which a singly-protonated Asp-Asp pair forms a hydrogen-bonded carboxyl-carboxylate clamp that clasps onto a charged Lys side chain. This polar motif was also supported by density functional theory and a Protein Data Bank–wide search. In contrast, the helices are dynamic at sites distal to the stable carboxyl-carboxylate clamp motif. Such a locally stable but globally dynamic structure is well suited to accommodate the sequence and structural variations in the transmembrane helices of multicomponent receptors, which mix and match subunits to create combinatorial functional diversity from a limited number of subunits. It also supports a signaling mechanism based on multisubunit clustering rather than propagation of rigid conformational changes through the membrane.

The CD3ζ adaptor structure determines functional differences between human and mouse CD16 Fc receptor signaling

“…The coordinates for the human CD3ζ transmembrane homodimer (ζ-ζ) were obtained from the NMR structure (PDB ID: 2HAC ; Call et al, 2006 ). The pair of membrane-embedded Asp residues necessary for assembly (D36 on each monomer) was monoprotonated with an overall charge of −1 based on previous findings ( Fong et al, 2021 ). All other residues were assigned their standard protonation states at pH 7.…”

Structural basis of γ chain family receptor sharing at the membrane level

“…S3) did not show any conserved small-xxx-small motif that mediates TMH dimerization in growth factor receptors ( 16 , 24 – 26 ). Nor did they show charged residues that mediate intramembrane assembly of activating immunoreceptors ( 27 – 29 ). Thus, pairing of the γc TMD with different ILR TMDs was mediated possibly by a previously unrecognized type of interaction.…”

ITAM‐based receptors in natural killer cells

“…Assembly is localized to the 2-to-1 motif, which retains a locally stable conformation. 5 In the absence of one of the three polar residues through mutation, the local conformation is disrupted, and assembly and therefore signaling are abrogated. 6,7 Despite the conformational stability of the 2-to-1 polar residue motif, a globally dynamic arrangement of the protein helices has been observed in the NKG2C-DAP12 receptor complex.…”

“…6,7 Despite the conformational stability of the 2-to-1 polar residue motif, a globally dynamic arrangement of the protein helices has been observed in the NKG2C-DAP12 receptor complex. 5 This globally dynamic arrangement provides insight into how DAP12, a highly promiscuous signaling subunit, is able to associate with so many ligand-binding subunits so long as they contain a basic residue in their transmembrane region. This finding also provides insight into how other adaptor molecules may accommodate multiple receptors with conserved, yet unique TM alpha helices.…”

“…This finding also provides insight into how other adaptor molecules may accommodate multiple receptors with conserved, yet unique TM alpha helices. 3,5 However, some multicomponent receptors do not rely on a 2-to-1 binding motif for assembly, as is the case for CD16. Instead, assembly in this receptor relies on a complex network of polar and nonpolar residues 8 that is highly sensitive to even very conservative mutations (discussed below).…”