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Figure 14: Diagrammatic representation of ID2 HLH structure
(A) Cartoon representation of the overall structure of ID2 HLH showing 2 monomer chains
(chain A in purple and chain B in brown) with α-helices labeled as α 1 and α2 linked by a loop
(green). Potassium ion is shown in grey.
(B) Top view (90° forward rotation) of ID2 HLH looking from the N-terminal downwards shows
an acetate molecule
(C) Acetate molecule likely contributed from the mother liquor of Potassium Acetate during
crystallization. 2Fo-Fc map contoured at 1.5σ. Cysteine residues are highlighted with the
distance between them at 15.9 Å to show the unlikely possibility of disulfide bond formation
(D) Potassium ion coordination site showing residues Lys47, Val50, Ile53, and Gln55 with 2Fo-
Fc map contoured at 1.5σ.

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The overall topology of the ID2 homodimer was similar to other bHLH-containing
proteins such as E47 and MYOD1 and conformed to the same parallel, 4-helix
bundle. The two main differences were the N-terminus and loop regions. The N-
terminus of bHLH-containing proteins contained an extended helix of basic residues
required to bind DNA; the density in this region was not interpretable in ID2 and was
likely disordered. In the loop region of both monomers was density that was too large
to be water. Based on the diameter and the orientation of the charged atoms, a
positive ion was modeled in the density. This was the first report of a loop ion for HLH
structures (Figure 15, grey sphere).
Figure 15: Cartoon representation of the crystal structure of ID2 at 2.1Å resolution showing the
positive loop ion and missing basic region.

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expected band location based on previous gel experiments). It was inferred that
failure to dimerize led to a failure to fold properly and hence, caused insolubility. This
showed that the hydrophobic core alone was not enough to hold the ID2 homodimer
together; that this hydrogen bond was critical to ID2’s structure. Secondarily, it
implied that the monomer was either not stable on its own or more prone to
degradation.
In addition to the inter-chain hydrogen bonds, there was one intra-chain hydrogen
bond at K61.cAα2 and N40.cAα1 in ID2 (Figure 16B). This interaction was only seen
in chain A of ID2 at 3.1Å and chain B of the ID3 (Q66 – N45) NMR structure at
around 3.8Å. Because the distances in the models were on the boundaries of a
hydrogen bond, it was thought to be unlikely to play a role in homodimerization.
However, the asapargine residue was highly conserved at a sequence level and
found in all Human IDs, E47 and Mouse E47 and NeuroD1 whereas the lysine in ID2
was a glutamine in ID3 at equivalent positions (Figure 19). Therefore K61 was
mutated in ID2 and Q66 was mutated in ID3 to alanines to test for effects on
homodimerization.