A cryptic phosphate-binding pocket on the SPFH domain of human stomatin that regulates a novel fibril-like self-assembly

Koki Kataoka; Shota Suzuki; Takeshi Tenno; Natsuko Goda; Emi Hibino; Atsunori Oshima; Hidekazu Hiroaki

doi:10.1016/j.crstbi.2022.05.002

A cryptic phosphate-binding pocket on the SPFH domain of human stomatin that regulates a novel fibril-like self-assembly

Curr Res Struct Biol. 2022 May 18:4:158-166. doi: 10.1016/j.crstbi.2022.05.002. eCollection 2022.

Authors

Koki Kataoka¹, Shota Suzuki², Takeshi Tenno^{1

3}, Natsuko Goda¹, Emi Hibino¹, Atsunori Oshima^{2

4}, Hidekazu Hiroaki^{1

3}

Affiliations

¹ Laboratory of Structural Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa-ku, Nagoya, Aichi, 464-8601, Japan.
² Cellular and Structural Physiology Institute (CeSPI), Nagoya University, Furocho, Chikusa-ku, Nagoya, Aichi, 464-8601, Japan.
³ BeCellBar LLC, Business Incubation Building, Nagoya University, Furocho, Chikusa-ku, Nagoya, Aichi, 464-8601, Japan.
⁴ Institute for Glyco-core Research (iGCORE), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.

Abstract

Human stomatin (hSTOM) is a component of the membrane skeleton of erythrocytes that maintains the membrane's shape and stiffness through interconnecting spectrin and actin. hSTOM is a member of the protein family that possesses a single stomatin/prohibitin/flotillin/HflK (SPFH) domain at the center of the molecule. Although SPFH domain proteins are widely distributed from archaea to mammals, the detailed function of the domain remains unclear. In this study, we first determined the solution structure of the SPFH domain of hSTOM (hSTOM(SPFH)) via NMR. The solution structure of hSTOM(SPFH) is essentially identical to the already reported crystal structure of the STOM SPFH domain (mSTOM(SPFH)) of mice, except for the existence of a small hydrophilic pocket on the surface. We identified this pocket as a phosphate-binding site by comparing its NMR spectra with and without phosphate ions. Meanwhile, during the conventional process of protein NMR analysis, we eventually discovered that hSTOM(SPFH) formed a unique solid material after lyophilization. This lyophilized hSTOM(SPFH) sample was moderately slowly dissolved in a physiological buffer. Interestingly, it was resistant to dissolution against the phosphate buffer. We then found that the lyophilized hSTOM(SPFH) formed a fibril-like assembly under electron microscopy. Finally, we succeeded in reproducing this fibril-like assembly of hSTOM(SPFH) using a centrifugal ultrafiltration device, thus demonstrating that the increased protein concentration may promote self-assembly of hSTOM(SPFH) into fibril forms. Our observations may help understand the molecular function of the SPFH domain and its involvement in protein oligomerization as a component of the membrane skeleton. (245 words).

Keywords: CD (circular dichroism), NMR (nuclear magnetic resonance); CSPs (chemical shift perturbations), CSTC (chemical shifts temperature coefficients); NOE (nuclear Overhauser effect), STOM (stomatin); PDB (Protein Data Bank), SPFH (stomatin/prohibitin/flotillin/HflK); TEM (transmission electron microscopy), r. m. s. d. (root mean square difference).