The N-terminal region of Ncb5or is predicted to be intrinsically disordered.
The amino acid sequence of the N-terminal region of human Ncb5or is shown in Figure 1B , and an alignment of this 50-residue polypeptide with all known Ncb5or examples in animals is included inFigure S1 . The alignment revealed an overall homology, which is weaker in the first 21 residues than the subsequent 29-residue stretch which is rich in Arg and Lys residues and contains five invariant residues (Lys25, Leu28, Gly31, Trp37 and Gly49). Trp37 is part of a highly conserved motif that has the sequence L34MDWIRL40 in mammalian orthologs.
The PONDR VL-XT program28-30predicts that the N-terminal region of Ncb5or is intrinsically disordered, in sharp contrast to the b5, b5R and CS domains (Figure S2A ). Secondary structure prediction algorithms generally gave inconsistent predictions for the N-terminal region, but several of them indicated a preference for α-helical structure for the L34MDWIRL40 sequence. This included PROF31 and PSIPRED32, 33 (Figure S2B ), as well as SPINE-X34 (not shown). The latter two ab initio algorithms (i.e. , those not utilizing homology modeling) have been shown to yield the most accurate predictions.35AGADIR36 , an algorithm that predicts helical content of peptides on the basis of helix/coil transition theory, also predicts some helical tendency for the L34MDWIRL40 sequence, albeit with a population of less than 10% at pH 7 and 25°C (Figure S2C ).