Highlights d A complex of STTs acts in sorting chloroplast Tat substrates to the thylakoids d Tat substrate activates the STT complex to undergo phase separation d Hcf106 reverses phase separation and facilitates cargo translocation

Tetrapyrroles, including haem and chlorophyll, play vital roles for various biological processes, such as respiration and photosynthesis, and their biosynthesis is critical for virtually all organisms. In photosynthetic organisms, magnesium chelatase (MgCh) catalyses insertion of magnesium into the centre of protoporphyrin IX, the branch-point precursor for both haem and chlorophyll, leading tetrapyrrole biosynthesis into the magnesium branch(1,2). This reaction needs a cooperated action of the three subunits of MgCh: the catalytic subunit ChlH and two AAA(+) subunits, ChlI and ChlD (refs 3-5). To date, the mechanism of MgCh awaits further elucidation due to a lack of high-resolution structures, especially for the ∼150 kDa catalytic subunit. Here we report the crystal structure of ChlH from the photosynthetic cyanobacterium Synechocystis PCC 6803, solved at 2.5 Å resolution. The active site is buried deeply inside the protein interior, and the surrounding residues are conserved throughout evolution. This structure helps to explain the loss of function reported for the cch and gun5 mutations of the ChlH subunit, and to provide the molecular basis of substrate channelling during the magnesium-chelating process. The structure advances our understanding of the holoenzyme of MgCh, a metal chelating enzyme other than ferrochelatase.

F-type ATP synthases produce nearly all of the ATP found in cells. The catalytic module F commonly comprises an αβ hexamer surrounding a γ/ε stalk. However, it is unclear how these subunits assemble to form a catalytic motor. In this work, we identified and characterized a chloroplast protein that interacts with the CFβ, γ, and ε subunits of the chloroplast ATP synthase and is required for assembly of its F module. We named this protein BIOGENESIS FACTOR REQUIRED FOR ATP SYNTHASE1 (BFA1) and determined its crystal structure at 2.8-Å resolution. BFA1 is comprised primarily of two interacting β-barrels that are oriented nearly perpendicularly to each other. The contact region between BFA1 and the CFβ and γ subunits was further mapped by yeast two-hybrid assays. An in silico molecular docking analysis was performed and revealed close fitting contact sites without steric conflicts between BFA1 and CFβ/γ. We propose that BFA1 acts mainly as a scaffold protein promoting the association of a CFα/β heterodimer with CFγ. The subsequent assembly of other CFα/β heterodimers may shift the position of the CFγ subunit to complete assembly of the CF module. This CF assembly process is likely to be valid for other F-type ATP synthases, as their structures are highly conserved.

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A structural study of A. thaliana glutamate-1-semialdehyde-2,1-aminomutase (GSAM) has revealed asymmetry in cofactor binding as well as in the gating-loop orientation, which supports the previously proposed negative cooperativity between monomers of GSAM.

The high temperature requirement A (HtrA) proteases (also termed Deg proteases) play important roles in diverse organisms by regulating protein quality and quantity. One of the 16 Arabidopsis homologs, Deg9, is located in the nucleus where it modulates cytokinin- and light-mediated signalling via degrading the ARABIDOPSIS RESPONSE REGULATOR 4 (ARR4). To uncover the structural features underlying the proteolytic activity of Deg9, we determined its crystal structure. Unlike the well-established trimeric building block of HtrAs, Deg9 displays a novel octameric structure consisting of two tetrameric rings that have distinct conformations. Based on the structural architecture, we generated several mutant variants of Deg9, determined their structure and tested their proteolytic activity towards ARR4. The results of the structural and biochemical analyses allowed us to propose a model for a novel mechanism of substrate recognition and activity regulation of Deg9. In this model, protease activation of one tetramer is mediated by en-bloc reorientation of the protease domains to open an entrance for the substrate in the opposite (inactive) tetramer. This study provides the structural basis for understanding how the levels of nuclear signal components are regulated by a plant protease.

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