*Article* **Natural Mutations Affect Structure and Function of gC1q Domain of Otolin-1**

**Rafał Hołubowicz \* , Andrzej Ozyhar ˙ and Piotr Dobryszycki \***

Department of Biochemistry, Molecular Biology and Biotechnology, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeze Wyspia ´ ˙ nskiego 27, 50-370 Wrocław, Poland; andrzej.ozyhar@pwr.edu.pl

**\*** Correspondence: rafal.holubowicz@pwr.edu.pl (R.H.); piotr.dobryszycki@pwr.edu.pl (P.D.); Tel.: +48-71-320-63-34 (R.H.); +48-71-320-63-32 (P.D.)

**Abstract:** Otolin-1 is a scaffold protein of otoliths and otoconia, calcium carbonate biominerals from the inner ear. It contains a gC1q domain responsible for trimerization and binding of Ca2+. Knowledge of a structure–function relationship of gC1q domain of otolin-1 is crucial for understanding the biology of balance sensing. Here, we show how natural variants alter the structure of gC1q otolin-1 and how Ca2+ are able to revert some effects of the mutations. We discovered that natural substitutions: R339S, R342W and R402P negatively affect the stability of apo-gC1q otolin-1, and that Q426R has a stabilizing effect. In the presence of Ca2+, R342W and Q426R were stabilized at higher Ca2+ concentrations than the wild-type form, and R402P was completely insensitive to Ca2+. The mutations affected the self-association of gC1q otolin-1 by inducing detrimental aggregation (R342W) or disabling the trimerization (R402P) of the protein. Our results indicate that the natural variants of gC1q otolin-1 may have a potential to cause pathological changes in otoconia and otoconial membrane, which could affect sensing of balance and increase the probability of occurrence of benign paroxysmal positional vertigo (BPPV).

**Keywords:** analytical ultracentrifugation; C1q; calcium binding proteins; circular dichroism; genetic variation; otoconia; otolin-1; OTOL1; site-directed mutagenesis; thermal shift assay
