Patrick Koenig

The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures.

Transport of precursor proteins across chloroplast membranes involves the GTPases Toc33/34 and Toc159 at the outer chloroplast envelope. The small GTPase Toc33/34 can homodimerize, but the regulation of this interaction has remained elusive. We show that dimerization is independent of nucleotide loading state, based on crystal structures of dimeric Pisum sativum Toc34 and monomeric Arabidopsis thaliana Toc33. An arginine residue is--in the dimer--positioned to resemble a GAP arginine finger. However, GTPase activation by dimerization is sparse and active site features do not explain catalysis, suggesting that the homodimer requires an additional factor as coGAP. Access to the catalytic center and an unusual switch I movement in the dimeric structure support this finding. Potential binding sites for interactions within the Toc translocon or with precursor proteins can be derived from the structures.

Conserved Properties of Polypeptide Transport-associated (POTRA) Domains Derived from Cyanobacterial Omp85

Proteins of the Omp85 family are conserved in all kingdoms of life. They mediate protein transport across or protein insertion into membranes and reside in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Omp85 proteins contain a C-terminal transmembrane β-barrel and a soluble N terminus with a varying number of polypeptide-transport-associated or POTRA domains. Here we investigate Omp85 from the cyanobacterium Anabaena sp. PCC 7120. The crystallographic three-dimensional structure of the N-terminal region shows three POTRA domains, here named P1 to P3 from the N terminus. Molecular dynamics simulations revealed a hinge between P1 and P2 but in contrast show that P2 and P3 are fixed in orientation. The P2-P3 arrangement is identical as seen for the POTRA domains from proteobacterial FhaC, suggesting this orientation is a conserved feature. Furthermore, we define interfaces for protein-protein interaction in P1 and P2. P3 possesses an extended loop unique to cyanobacteria and plantae, which influences pore properties as shown by deletion. It now becomes clear how variations in structure of individual POTRA domains, as well as the different number of POTRA domains with both rigid and flexible connections make the N termini of Omp85 proteins versatile adaptors for a plentitude of functions.

Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33

GTPases act as molecular switches to control many cellular processes, including signalling, protein translation and targeting. Switch activity can be regulated by external effector proteins or intrinsic properties, such as dimerization. The recognition and translocation of pre-proteins into chloroplasts [via the TOC/TIC (translocator at the outer envelope membrane of chloroplasts/inner envelope membrane of chloroplasts)] is controlled by two homologous receptor GTPases, Toc33 and Toc159, whose reversible dimerization is proposed to regulate translocation of incoming proteins in a GTP-dependent manner. Toc33 is a homodimerizing GTPase. Functional analysis suggests that homodimerization is a key step in the translocation process, the molecular functions of which, as well as the elements regulating this event, are largely unknown. In the present study, we show that homodimerization reduces the rate of nucleotide exchange, which is consistent with the observed orientation of the monomers in the crystal structure. Pre-protein binding induces a dissociation of the Toc33 homodimer and results in the exchange of GDP for GTP. Thus homodimerization does not serve to activate the GTPase activity as discussed many times previously, but to control the nucleotide-loading state. We discuss this novel regulatory mode and its impact on the current models of protein import into the chloroplast.

ON THE SIGNIFICANCE OF TOC-GTPASE HOMODIMERS *

Precursor protein translocation across the outer chloroplast membrane depends on the action of the Toc complex, containing GTPases as recognizing receptor components. The G domains of the GTPases are known to dimerize. In the dimeric conformation an arginine contacts the phosphate moieties of bound nucleotide in trans. Kinetic studies suggested that the arginine in itself does not act as an arginine finger of a reciprocal GTPase-activating protein (GAP). Here we investigate the specific function of the residue in two GTPase homologues. Arginine to alanine replacement variants have significantly reduced affinities for dimerization compared with wild-type GTPases. The amino acid exchange does not impact on the overall fold and nucleotide binding, as seen in the monomeric x-ray crystallographic structure of the Arabidopsis Toc33 arginine-alanine replacement variant at 2.0Å. We probed the catalytic center with the transition state analogue GDP/AlFx using NMR and analytical ultracentrifugation. AlFx binding depends on the arginine, suggesting the residue can play a role in catalysis despite the non-GAP nature of the homodimer. Two non-exclusive functional models are discussed: 1) the coGAP hypothesis, in which an additional factor activates the GTPase in homodimeric form; and 2) the switch hypothesis, in which a protein, presumably the large Toc159 GTPase, exchanges with one of the homodimeric subunits, leading to activation.

Structure and Conservation of the Periplasmic Targeting Factor Tic22 Protein from Plants and Cyanobacteria

Background: Although Tic22 is involved in protein import into chloroplasts, the function in cyanobacteria is unknown. Results: Cyanobacterial Tic22 is required for OM biogenesis, shares structural features with chaperones, and can be substituted by plant Tic22. Conclusion: Tic22, involved in outer membrane biogenesis, is functionally conserved in cyanobacteria and plants. Significance: The findings are important for the understanding of periplasmic protein transport. Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration

Background: Engineering Fabs with high affinity toward two distinct antigens is challenged by the competing constraints of a shared binding surface. Results: Deep mutational scan unveiled the sequences of Fabs with sub-nanomolar affinity for two angiogenic targets. Conclusion: Fabs potent against two structurally unrelated targets were discovered. Significance: Efficacious generation of dual action Fab expands the therapeutic potential of Fab molecules in ocular indications and beyond. The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies.

pH Sensitivity of the GTPase Toc33 as a Regulatory Circuit for Protein Translocation into Chloroplasts

The properties of membrane-embedded GTPases are investigated to understand translocation of preprotein across the outer envelope of chloroplasts. The homo- and heterodimerization events of the GTPases had been established previously. We show that the hydrolytic activity of the GTPase Toc33 is pH insensitive in the homodimeric conformation but has a bell-shaped pH optimum in the monomeric conformation. Further, Toc33 GTPase homodimerization and protein translocation into chloroplasts are pH sensitive as well. pH sensitivity might serve to regulate translocation; alternatively, the documented pH sensitivity might reflect a mechanistic requirement for GTPase silencing during translocation as the GTPase switches between homo- and heterodimeric conformations.

Protein export in cyanobacteria - a model for organelle protein transport

The insertion of proteins into and the transport across outer membranes in cyanobacteria has a strong semblance to protein import into eukaryotic organelles, with a swap in directionality [1]. Specific transporters mediate the transfer across and the insertion into bio-membranes. We focus on the study of beta-barrel proteins that reside in the outer membranes and periplasmatic factors that mediate protein recognition and transport. The beta barrel proteins have a varying number of socalled POTRA domains (POlypeptideTRansport-Associated), with inherent flexibility between individual POTRA domains [2]. These recognise target proteins and facilitate protein transport through the beta barrel [2], [3]. Chaperones similar to protobacterial SurA and Skp/DegP assist cyanobacterial protein transport through the periplasm. The 3D structure of the cyanobacterial chaperone Tic22 has a “butterfly” shape revealing a repeat likely caused by gene duplication [4], [5]. Four helices point orthogonal at each other, adding up their dipole moments in a central cavity. The surface of the structure is dotted with hydrophobic pockets in which we identified bound solvent molecules. These likely represent binding sites for protein substrates. We demonstrate that Tic22 is present in the cyanobaterial periplasm as well as in thylakoids, and it can be functionally replaced by knock-in of a plant orthologue [4]. In the apicoplast organelle of unicellular parasites such as Plasmodium and Toxoplasma, Tic22 is essential for parasite survival and protein import into the apicoplast stroma [5]. The structural clues together with the functional data suggest that Tic22 can have a function in both, protein import or protein insertion, depending on the organism where it is found. The protein is conserved in bacteria, plants, and unicellular organisms and links these protein transporters to a common ancestry.