Allosteric Activation of a Spring-Loaded Natriuretic Peptide Receptor Dimer by Hormone Xiao-lin He, Dar-chone Chow, Monika M. Martick, and K. Christopher Garcia

Supplementary Material

Supplemental Figure 1. Electron density of the NPR-C structures, and the bound CNP hormone. (A) SIGMAA-weighted omit map (2Fo-Fc) of a portion of the dimer interface between the NH₂-terminal domains of the NPR-C complex at 2.0 Å resolution. (B) SIGMAA-weighted omit map of the N-linked glycan at Asparagine-248 on the unliganded NPR-C at 2.9 Å. (C) SIGMAA-weighted omit map of the single bound CNP peptide conformation in two orientations at 2.0 Å from the side (left) and two "end-on" views looking from the top down (top) and the bottom up (bottom). The twofold symmetry of the peptide electron density is clear in all three views, as is the identical unique conformation of the peptide in both orientations.

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Supplemental Figure 2. Comparison of NPR-A and NPR-C dimers. (A) A crystal packing diagram [with the program O (40)] of the unliganded rat NPR-A structure is shown indicating the "tail-to-tail" dimer initially chosen to accommodate a 2:2 stoichiometry. However, the physiologically-relevant NPR-A dimer consistent with a 1:2 stoichiometry is in the same crystal lattice and is identical to that seen in human NPR-C. (B) The physiologically relevant dimer of NPR-A is aligned by the membrane-proximal domains to NPR-C, showing the identical topology. As for unliganded NPR-C, hormone binding to this NPR-A dimer will require closing of the interdimer gap.

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Supplemental Table 1. Site I and Site II contact surfaces (top) and hydrogen bonds (bottom) in the NPR-C/CNP interfaces. Buried surface area was calculated with a 1.4 Å probe radius.
Site I (Total buried surface 1346 Å2)					Site II (Total buried surface 1018 Å2)
CNP	Neighboring NPR-C (I) residues (<4.5 Å)				Neighboring NPR-C ( II) residues (<4.5 Å)
Gly5	I188
Cys6	Y159*, I188
Phe7	Y159*, Y169, L172, E173*, H176, I188
Gly8	H176
Leu9	H176, E177, Q180
Lys10					K163
Leu11	Y169				K163
Asp12	G118*, H121, Y126*
Arg13	R100*, E125				N65, Y93*, K163
Ile14	Y93*, R166*, Y169, F170*
Gly15	Y93*				Y93*
Ser16					Y93*, A96*, R100*
Met17	Y93*				A96*, P97, R100*, G118*, F119*, Y126*
Ser18	K163
Gly19					E173*
Leu20					Y169*
Gly21					Y169*
Cys22
*conserved across NPR family. similar hydrophobic or aromatic residues.
Hydrogen bonds between NPR-C and CNP in site I and site II of the complex
Site I atoms					Site II atoms
NPR-C ( I )	CNP	Distance (Å)			NPR-C ( II )	CNP	Distance (Å)
E125 O1	R13 N2	3.1			R100* N1	Ser 16 O	3.2
E125 O2	R13 N1	2.7			Y169# OH	Gly21 O	3.0
H176 N2	G 8 N	3.1			E173* O2	Leu 20 N	3.1
* conserved across NPR family.