The impact of disease mutations on homodimer formation of Rpgrip1l CC1/CC2 or Rpgrip1 CC12 (A) The mutation sites in Rpgrip1l CC12 found in the Clinvar database are mapped onto the structures and highlighted with red spheres. (B) SEC-MALS results showing that R47H or L57 M/F had little effect on the homodimer formation of Rpgrip1l CC1. The measured molecular weights are expressed as Mn (Gx%). (C) SEC-MALS result showing that L129H can disrupt dimerization of Trx-Rpgrip1l CC2. The measured molecular weights are expressed as M n (Gx%). (D) SEC-MALS results showing that R43P and R43W resulted in different oligomeric states of GB1-human Rpgrip1 CC12. The measured molecular weights are expressed as M n (Gx%).

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... total, 18 missense mutations were located in the CC1 region and 6 in the CC2 region of Rpgrip1l (Table S1). We mapped these mutational sites on the structures and found that three of them (R47H, L57 M/F in CC1, and L129H in CC2) are in the dimer interface, although the majority of them are not ( Figure 6A). iScience Article For the Rpgrip1l CC1 region, as described above, R47 forms a hydrogen bond with the backbone of V45 0 , and L57 is one of the key hydrophobic residues at the d position. ...

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... substituting L57 with another two hydrophobic residues may not disrupt the dimer. As a result, R47H, L57F, and L57M mutations did not alter the homodimer state of GB1-tagged Rpgrip1l CC1 ( Figure 6B). For the CC2 region, L129, which has been shown to be critical for dimer formation, was mutated to His in some patients with JBTS or MKS. ...

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... the CC2 region, L129, which has been shown to be critical for dimer formation, was mutated to His in some patients with JBTS or MKS. The SEC-MALS result demonstrated that this variant was a monomer ( Figure 6C). Since both CC1 and CC2 alone can dimerize, Rpgrip1l CC12 with any one of these mutations can still form dimers (Fig- ure S5A), although the affinity might be lower. ...

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... R43G and R43Q of hRP1 barely impacted the dimerization. However, the R43P mutation broke the CC1 dimer into a monomer, whereas the R43W mutation led to heterogeneous nonspecific oligomers with much higher molecular weights ( Figure 6D). The CD results demonstrated that the helical structures or the stability remain nearly unchanged with the R43G, R43Q, or R43W mutants, and R43P obviously decreased the helical content ( Figure S6). ...

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... the R43P mutation broke the CC1 dimer into a monomer, whereas the R43W mutation led to heterogeneous nonspecific oligomers with much higher molecular weights ( Figure 6D). The CD results demonstrated that the helical structures or the stability remain nearly unchanged with the R43G, R43Q, or R43W mutants, and R43P obviously decreased the helical content ( Figure S6). ...