Figure 5. 3D structure reveals an inhibitory ARM allosteric NAD+ binding site.

(A) Selected representation of 2D class averages used for the 3D reconstruction of hSARM1^E642Q supplemented with 5 mM of NAD+. The number of particles that were included in each class average is indicated. (B) Color coded (as in Figure 1A) protein model docked in a transparent 2.7 Å cryo-EM density map (gray). (C) When compared to the GraFix-ed map, without NAD+ supplement (left), an extra density appears at the ‘ARM horns’ region in the NAD+ supplemented map (middle, right). The extra density is rendered in green and an NAD+ molecule is fitted to it. The NAD+ is surrounded by three structural elements, as indicated on the right panel. The NAD+ directly interacts with the surrounding residues: the nicotinamide moiety is stacked onto the W103 side chain rings; the following ribose with L152 and H190; R110 forms salt bridge with phosphate alpha (proximal to the nicotinamide), while the beta phosphate (distal to the nicotinamide) forms salt bridges with R110 and R157. The map density at the distal ribose and adenosine moieties is less sharp, but clearly involves interactions with the ARM² R322, G323, and D326. In this way, activation by nicotinamide mononucleotide, that lacks the distal phosphate, ribose, and adenosine, can be explained by binding to ARM¹ while preventing the bridging interactions with ARM². (D) Toxicity of the hSARM1 construct and mutants in HEK293F cells. The cells were transfected with hSARM1 expression vectors, as indicated. Viable cells were counted 48 (bars in gray) and 72 (bars in black stripes) hr post-transfection. Moderate reduction in cell viability due to ectopic expression of hSARM1^w.t. becomes apparent 72 hr after transfection, when compared with the NADase attenuated hSARM1^E642Q, while the ‘delARM’ construct marks a constitutive activity that brings about almost complete cell death after 3 days. Mutations at the ARM¹ α5–α6 and ARM¹–ARM² loops induce cell death at a similar level as the ‘delARM’ construct, while the control mutations and W103A did not show increased activity (three biological repeats, Student’s t-test; ***p<0.001; *p<0.05; n.s: no significance).

Figure 5—figure supplement 1. Cartoon model and density map (in transparent gray) of the NAD+ supplemented hSARM1.

Color code is as in Figure 1A. (A) A zoom-in bottom view with indication of the ‘ARM horns’ region. NAD+ densities are in green. Note the ARM domain interactions with neighboring SAM and ARM domains. (B) A zoom-in view of the TIR domain docking onto two neighboring ARM domains. The NAD+ binding cleft and BB loop are indicated. (C) Density map (in mesh) showing a close-up view of isolated segments of the ARM, SAM, and TIR sections. The backbone and side chains are represented as sticks and colored in yellow, blue, and red, respectively, as in Figure 1A.

Figure 5—figure supplement 2. Zooming-in on the ARM allosteric NAD+ binding site.

(A, B) The principle structural rearrangements in the ARM domain are presented as simplified illustration based on a comparison between the GraFix-ed (A) and the NAD+ supplemented (B) SARM1 cryo-EM maps. The ARM¹ and ARM² subdomains are depicted in yellow and orange, respectively, and the ARM¹ α2–α3 loop and ARM¹ and ARM² are highlighted. (C) 2D Ligplot (Wallace et al., 1995) diagram details the hydrogen bonds, salt bridges, and hydrophobic interactions between NAD+ and SARM1, where ARM¹ residues are colored in yellow, and ARM² residues in orange. The nicotinamide mononucleotide (NMN) moiety of NAD+ is outlined by a gray line drawing. According to this analysis, the NMN moiety mostly interacts (but not only) with ARM¹ residues: W103, L104, E149, Q150, L152, V153, H190, and has a partial interaction with R110. S316 and D317 of ARM² also interact with the NMN moiety. The adenosine half of NAD+ mostly interacts (but not only) with ARM² residues: Q320, G321, R322, G323, and D326. R110 and R157 of ARM¹ also interact with the adenosine half.