Operon flv4-flv2 provides cyanobacterial photosystem II with flexibility of electron transfer.

Synechocystis sp PCC 6803 has four genes encoding flavodiiron proteins (FDPs; Flv1 to Flv4). Here, we investigated the flv4-flv2 operon encoding the Flv4, Sll0218, and Flv2 proteins, which are strongly expressed under low inorganic carbon conditions (i.e., air level of CO(2)) but become repressed at elevated CO(2) conditions. Different from FDP homodimers in anaerobic microbes, Synechocystis Flv2 and Flv4 form a heterodimer. It is located in cytoplasm but also has a high affinity to membrane in the presence of cations. Sll0218, on the contrary, resides in the thylakoid membrane in association with a high molecular mass protein complex. Sll0218 operates partially independently of Flv2/Flv4. It stabilizes the photosystem II (PSII) dimers, and according to biophysical measurements opens up a novel electron transfer pathway to the Flv2/Flv4 heterodimer from PSII. Constructed homology models suggest efficient electron transfer in heterodimeric Flv2/Flv4. It is suggested that Flv2/Flv4 binds to thylakoids in light, mediates electron transfer from PSII, and concomitantly regulates the association of phycobilisomes with PSII. The function of the flv4-flv2 operon provides many ß-cyanobacteria with a so far unknown photoprotection mechanism that evolved in parallel with oxygen-evolving PSII.

Structure of collagen receptor integrin α(1)I domain carrying the activating mutation E317A.

We have analyzed the structure and function of the integrin α(1)I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α(1)I C139S/E317A was a higher avidity collagen binder than α(1)I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α(1)I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α(2)I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α(2)I structure, has not changed its position in the activated α(1)I variant. During the integrin activation, Glu(335) on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the ß(1) subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu(335). This indicates that the stabilization of helix 7 into its downward position is not required if the α(1) MIDAS is already open. To conclude, the activated α(1)I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg(287)-Glu(317) ion pair has just broken during the integrin activation.

Characterization of the substrate-binding PotD subunit in Synechocystis sp. strain PCC 6803.

The potD gene encodes the bacterial substrate-binding subunit of the polyamine transport system. The uptake system, which belongs to the ABC transporters, has been characterized in Escherichia coli, but it has not been previously studied in cyanobacteria. Although the overall sequence identity between Synechocystis sp. strain PCC 6803 (hereafter Synechocystis) PotD and Escherichia coli PotD is 24%, the ligand-binding site in the constructed homology model of Synechocystis PotD is well conserved. The conservation of the five polyamine-binding residues (Asp206, Glu209, Trp267, Trp293, and Asp295 in Synechocystis PotD) between these two species indicated polyamine-binding capacity for Synechocystis PotD. The Synechocystis potD gene is functional and its expression is under environmental regulation at transcriptional as well as post-transcriptional levels. Furthermore, an in vitro binding assay with the purified recombinant PotD protein demonstrated that the Synechocystis PotD protein is able to bind polyamines and favors spermidine over putrescine. Finally, we confirmed that Synechocystis PotD plays a physiological role in the uptake of polyamines in vivo using a constructed Synechocystis potD-disruption mutant.

Transcriptional regulation and structural modeling of the FutC subunit of an ABC-type iron transporter in Synechocystis sp. strain PCC 6803.

The futC gene encodes a subunit of an ATP-binding cassette (ABC)-type iron transporter in Synechocystis sp. strain PCC 6803. In the present study, we have focused on the environmental regulation of futC transcription in the model organism Synechocystis sp. strain PCC 6803 and, moreover, studied the transcriptional regulation of the other transporter subunits, futA1, futA2 and futB. The steady-state amounts of the futA1, futA2, futB and futC transcripts were regulated under several conditions studied including darkness, temperature, alternative nitrogen source, salt and osmotic stresses and iron deficiency. Transcription of all subunits of the FutABC-iron transporter seems to be under similar regulation, which, according to our results, may also apply to genes encoding subunits of other transporters in Synechocystis. The sequence alignment, including sequences from six different organisms, revealed the conserved nature of FutC. Based on the sequence alignment and the structural model of FutC, the monomer consists of a nucleotide-binding domain (NBD) and a regulatory domain. The NBD is well conserved indicating completely functional ATP binding.

Effects of conformational activation of integrin alpha 1I and alpha 2I domains on selective recognition of laminin and collagen subtypes.

Collagen receptor integrins alpha 1 beta 1 and alpha 2 beta 1 can selectively recognize different collagen subtypes. Here we show that their alpha I domains can discriminate between laminin isoforms as well: alpha 1I and alpha 2I recognized laminin-111, -211 and -511, whereas their binding to laminin-411 was negligible. Residue Arg-218 in alpha1 was found to be instrumental in high-avidity binding. The gain-of-function mutation E318W makes the alpha 2I domain to adopt the "open" high-affinity conformation, while the wild-type alpha 2I domain favors the "closed" low-affinity conformation. The E318W mutation markedly increased alpha 2I domain binding to the laminins (-111, -211 and -511), leading us to propose that the activation state of the alpha 2 beta 1 integrin defines its role as a laminin receptor. However, neither wild-type nor alpha 2IE318W domain could bind to laminin-411. alpha 2IE318W also bound tighter to all collagens than alpha 2I wild-type, but it showed reduced ability to discriminate between collagens I, IV and IX. The corresponding mutation, E317A, in the alpha 1I domain transformed the domain into a high-avidity binder of collagens I and IV. Thus, our results indicate that conformational activation of integrin alpha 1I and alpha 2I domains leads to high-avidity binding to otherwise disfavored collagen subtypes.