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Conserved Residues in the C-Terminal Domain Affect the Structure and Function of CYP38 in Arabidopsis

ABSTRACT: Arabidopsis cyclophilin38 (CYP38) is a thylakoid lumen protein critial for PSII assembly and maintenance, and its C-terminal region serves as the target binding domain. We hypothesized that four conserved residues (R290, F294, Q372, and F374) in the C-terminal domain are critical for the structure and function of CYP38. In yeast two-hybrid and protein pull-down assays, CYP38s with single-sited mutations (R290A, F294A, Q372A, or F374A) did not interact with the CP47 E-loop as the wild-type CYP38. In contrast, CYP38 with the R290A/F294A/Q372A/F374A quadruple mutation could bind the CP47 E-loop. Gene transformation analysis showed that the quadruple mutation prevented CYP38 to efficiently complement the mutant phenotype of cyp38. The C-terminal domain half protein with the quadruple mutation, like the wild-type one, could interact with the N-terminal domain or the CP47 E-loop in vitro. The cyp38 plants expressing CYP38 with the quadruple mutation showed a similar BN-PAGE profile as cyp38, but distinct from the wild type. The CYP38 protein with the quadruple mutation associated with the thylakoid membrane less efficiently than the wild-type CYP38. We concluded that these four conserved residues are indispensable as changes of all these residues together resulted in a subtle conformational change of CYP38 and reduced its intramolecular N-C interaction and the ability to associate with the thylakoid membrane, thus impairing its function in chloroplast.

SUBMITTER: Shi L

PROVIDER: S-EPMC7959726 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Project description:Epidermal cells of dark-grown plant seedlings reorient their cortical microtubule arrays in response to blue light from a net lateral orientation to a net longitudinal orientation with respect to the long axis of cells. The molecular mechanism underlying this microtubule array reorientation involves katanin, a microtubule severing enzyme, and a plant-specific microtubule associated protein called SPIRAL2. Katanin preferentially severs longitudinal microtubules, generating seeds that amplify the longitudinal array. Upon severing, SPIRAL2 binds nascent microtubule minus ends and limits their dynamics, thereby stabilizing the longitudinal array while the lateral array undergoes net depolymerization. To date, no experimental structural information is available for SPIRAL2 to help inform its mechanism. To gain insight into SPIRAL2 structure and function, we determined a 1.8 Å resolution crystal structure of the Arabidopsis thaliana SPIRAL2 C-terminal domain. The domain is composed of seven core α-helices, arranged in an α-solenoid. Amino-acid sequence conservation maps primarily to one face of the domain involving helices α1, α3, α5, and an extended loop, the α6-α7 loop. The domain fold is similar to, yet structurally distinct from the C-terminal domain of Ge-1 (an mRNA decapping complex factor involved in P-body localization) and, surprisingly, the C-terminal domain of the katanin p80 regulatory subunit. The katanin p80 C-terminal domain heterodimerizes with the MIT domain of the katanin p60 catalytic subunit, and in metazoans, binds the microtubule minus-end factors CAMSAP3 and ASPM. Structural analysis predicts that SPIRAL2 does not engage katanin p60 in a mode homologous to katanin p80. The SPIRAL2 structure highlights an interesting evolutionary convergence of domain architecture and microtubule minus-end localization between SPIRAL2 and katanin complexes, and establishes a foundation upon which structure-function analysis can be conducted to elucidate the role of this domain in the regulation of plant microtubule arrays.

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Conserved Residues in the C-Terminal Domain Affect the Structure and Function of CYP38 in Arabidopsis

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