Raphael Gasper

The retinitis pigmentosa 2 gene product is a GTPase-activating protein for Arf-like 3

The retinitis pigmentosa 2 (RP2) gene is responsible for a particular variant of X chromosome–linked eye disease. Previously, RP2 was shown to bind the GTP form of the small G protein Arf-like 3 (Arl3), thus qualifying as an effector. Here we present the Arl3–GppNHp–RP2 complex structure, which shows features resembling complexes with GTPase-activating proteins (GAPs). Biochemical analysis showing a 90,000-fold stimulation of the GTPase reaction together with the structure of an Arl3–GDP–AlF4−–RP2 transition state complex showed that RP2 is an efficient GAP for Arl3, with structural features similar to other GAPs. Furthermore, the effect of mutations in patients with retinitis pigmentosa correlated with their effect on catalysis, in particular the mutation of the arginine finger of RP2. The cognate G protein–GAP pair is conserved in yeast as Cin4–Cin2, and the ability of RP2 to act as a GAP can be correlated with its ability to complement a CIN2-deletion phenotype.

Structural insights into HypB, a GTP-binding protein that regulates metal binding.

HypB is a prokaryotic metal-binding guanine nucleotide-binding protein that is essential for nickel incorporation into hydrogenases. Here we solved the x-ray structure of HypB from Methanocaldococcus jannaschii. It shows that the G-domain has a different topology than the Ras-like proteins and belongs to the SIMIBI (after Signal Recognition Particle, MinD and BioD) class of NTP-binding proteins. We show that HypB undergoes nucleotide-dependent dimerization, which is apparently a common feature of SIMIBI class G-proteins. The nucleotides are located in the dimer interface and are contacted by both subunits. The active site features residues from both subunits arguing that hydrolysis also requires dimerization. Two metal-binding sites are found, one of which is dependent on the state of bound nucleotide. A totally conserved ENV/IGNLV/ICP motif in switch II relays the nucleotide binding with the metal ionbinding site. The homology with NifH, the Fe protein subunit of nitrogenase, suggests a mechanistic model for the switch-dependent incorporation of a metal ion into hydrogenases.

The role of the conserved switch II glutamate in guanine nucleotide exchange factor-mediated nucleotide exchange of GTP-binding proteins.

Guanine nucleotide exchange factors (GEFs) regulate the activity of small G proteins by catalysing the intrinsically slow exchange of GDP for GTP. The mechanism involves the formation of trimeric G protein-nucleotide-GEF complexes, followed by the release of nucleotide to form stable binary G protein-GEF complexes. A number of structural studies of G protein-GEF complexes have shown large structural changes induced in the nucleotide binding site. Together with a recent structure of a trimeric complex, these studies have suggested not only some common principles but also large differences in detail in the GEF-mediated exchange reaction. Several structures suggested that a glutamic acid residue in switch II, which is part of the DxxGQE motif and highly conserved in Ras-like G proteins, might have a decisive mechanistic role in GEF-mediated nucleotide exchange reactions. Here we show that mutation of the switch II glutamate to Ala severely impairs GEF-catalysed nucleotide exchange in most, but not all, Ras family G proteins, explaining its high sequence conservation. The residue determines the initial approach of GEF to the nucleotide-loaded G protein and does not appreciably affect the formation of a binary nucleotide-free complex. Its major effect thus appears to be the removal of the P-loop lysine from its interaction with the nucleotide.

RasGEF1A and RasGEF1B are guanine nucleotide exchange factors that discriminate between Rap GTP‐binding proteins and mediate Rap2‐specific nucleotide exchange

The highly conserved RasGEF1 family of proteins contain a C‐terminal CDC25‐Ras exchange motif domain and an N‐terminal RasGEF‐N domain, and are of unknown function and specificity. Using purified RasGEF1A and RasGEF1B proteins, as well as Ras family proteins, we established that RasGEF1A and RasGEF1B function as very specific exchange factors for Rap2, a member of the Rap subfamily of Ras‐like G‐proteins. They do not act on Rap1 or other members of the Ras subfamily. Although Rap2 was implicated in the regulation of cell adhesion, the establishment of cell morphology, and the modulation of synapses in neurons, no specific guanine nucleotide exchange factor for Rap2 was previously identified. Using reciprocal site‐directed mutagenesis, we analyzed residues that allow RasGEF1 proteins to discriminate between Rap1 and Rap2, and we were able to identify Phe39 in the switch I region of Rap2 as a specificity residue. Mutation of the corresponding Ser39 in Rap1 changed the specificity and allowed the nucleotide exchange of Rap1(S39F) to be stimulated by RasGEF1B.

Insights into the Biosynthesis and Assembly of Cryptophycean Phycobiliproteins

Background: Cryptophytes like Guillardia theta utilize soluble phycobiliproteins for light-harvesting. Results: Guillardia theta adopted phycoerythrobilin biosynthesis from cyanobacteria, and the phycobiliprotein lyase GtCPES provides structural requirements for transfer of this chromophore to a specific cysteine residue of the apophycobiliprotein. Conclusion: Phycobiliprotein synthesis in Guillardia theta combines proven and novel components. Significance: Results provide a better understanding of the evolution and function of unusual phycobiliproteins in cryptophytes. Phycobiliproteins are employed by cyanobacteria, red algae, glaucophytes, and cryptophytes for light-harvesting and consist of apoproteins covalently associated with open-chain tetrapyrrole chromophores. Although the majority of organisms assemble the individual phycobiliproteins into larger aggregates called phycobilisomes, members of the cryptophytes use a single type of phycobiliprotein that is localized in the thylakoid lumen. The cryptophyte Guillardia theta (Gt) uses phycoerythrin PE545 utilizing the uncommon chromophore 15,16-dihydrobiliverdin (DHBV) in addition to phycoerythrobilin (PEB). Both the biosynthesis and the attachment of chromophores to the apophycobiliprotein have not yet been investigated for cryptophytes. In this study, we identified and characterized enzymes involved in PEB biosynthesis. In addition, we present the first in-depth biochemical characterization of a eukaryotic phycobiliprotein lyase (GtCPES). Plastid-encoded HO (GtHo) was shown to convert heme into biliverdin IXα providing the substrate with a putative nucleus-encoded DHBV:ferredoxin oxidoreductase (GtPEBA). A PEB:ferredoxin oxidoreductase (GtPEBB) was found to convert DHBV to PEB, which is the substrate for the phycobiliprotein lyase GtCPES. The x-ray structure of GtCPES was solved at 2.0 Å revealing a 10-stranded β-barrel with a modified lipocalin fold. GtCPES is an S-type lyase specific for binding of phycobilins with reduced C15=C16 double bonds (DHBV and PEB). Site-directed mutagenesis identified residues Glu-136 and Arg-146 involved in phycobilin binding. Based on the crystal structure, a model for the interaction of GtCPES with the apophycobiliprotein CpeB is proposed and discussed.

GTPase activity of Di-Ras proteins is stimulated by Rap1GAP proteins

The Ras family is the largest and most diverse sub-group of Ras-like G proteins. This complexity is further increased by the high number of regulatory Guanine nucleotide Exchange Factors (GEFs) and GTPase activating proteins (GAPs) that target specific members of this subfamily. Di-Ras1 and Di-Ras2 are little characterized members of the Raslike sub-group with still unidentified regulatory and effector proteins. Here we determined the nucleotide binding properties of Di-Ras1/Di-Ras2. The above nanomolar affinity and the inability to react with members of the Cdc25 RasGEF family might suggest that activation does not require a GEF. We identified Rap1GAP1 and Rap1GAP2 as specific GTPase activating proteins of the Di-Ras family. Dual-specificity GAPs of the GAP1m family could not activate Di-Ras proteins, despite the presence of the required catalytic residue. Although DiRas proteins share GAPs with Rap G proteins, no common effectors could be identified in vitro.

Auxiliary metabolic genes- Distinct Features of Cyanophage-encoded T-type Phycobiliprotein Lyase θCpeT

Auxiliary metabolic genes (AMG) are commonly found in the genomes of phages that infect cyanobacteria and increase the fitness of the cyanophage. AMGs are often homologs of host genes, and also typically related to photosynthesis. For example, the ΦcpeT gene in the cyanophage P-HM1 encodes a putative phycobiliprotein lyase related to cyanobacterial T-type lyases, which facilitate attachment of linear tetrapyrrole chromophores to Cys-155 of phycobiliprotein β-subunits, suggesting that ΦCpeT may also help assemble light-harvesting phycobiliproteins during infection. To investigate this possibility, we structurally and biochemically characterized recombinant ΦCpeT. The solved crystal structure of ΦCpeT at 1.8-Å resolution revealed that the protein adopts a similar fold as the cyanobacterial T-type lyase CpcT from Nostoc sp. PCC7120 but overall is more compact and smaller. ΦCpeT specifically binds phycoerythrobilin (PEB) in vitro leading to a tight complex that can also be formed in Escherichia coli when it is co-expressed with genes encoding PEB biosynthesis (i.e. ho1 and pebS). The formed ΦCpeT·PEB complex was very stable as the chromophore was not lost during chromatography and displayed a strong red fluorescence with a fluorescence quantum yield of ΦF = 0.3. This complex was not directly able to transfer PEB to the host phycobiliprotein β-subunit. However, it could assist the host lyase CpeS in its function by providing a pool of readily available PEB, a feature that might be important for fast phycobiliprotein assembly during phage infection.

Dirigent Protein Mode of Action Revealed by the Crystal Structure of AtDIR6

Crystal structure analysis reveals the mode of substrate radical binding and indicates a previously unrecognized catalytic function for dirigent proteins during enantioselective pinoresinol formation. Dirigent proteins impart stereoselectivity to phenoxy radical coupling reactions in plants and, thus, play an essential role in the biosynthesis of biologically active natural products. This includes the regioselective and enantioselective coupling and subsequent cyclization of two coniferyl alcohol radicals to pinoresinol as the committed step of lignan biosynthesis. The reaction is controlled by dirigent proteins, which, depending on the species and protein, direct the reaction to either (+)- or (−)-pinoresinol. We present the crystal structure of the (−)-pinoresinol forming DIRIGENT PROTEIN6 (AtDIR6) from Arabidopsis (Arabidopsis thaliana) with data to 1.4 Å resolution. The structure shows AtDIR6 as an eight-stranded antiparallel β-barrel that forms a trimer with spatially well-separated cavities for substrate binding. The binding cavities are two lobed, exhibiting two opposing pockets, each lined with a set of hydrophilic and potentially catalytic residues, including essential aspartic acids. These residues are conserved between (+) and (−)-pinoresinol-forming DIRs and required for activity. The structure supports a model in which two substrate radicals bind to each of the DIR monomers. With the aromatic rings fixed in the two pockets, the propionyl side chains face each other for radical-radical coupling, and stereoselectivity is determined by the exact positioning of the side chains. Extensive mutational analysis supports a previously unrecognized function for DIRs in catalyzing the cyclization of the bis-quinone methide reaction intermediate to yield (+)- or (−)-pinoresinol.

Cloning, expression, crystallization and preliminary X‐ray studies of a superfolder GFP fusion of cyanobacterial Psb32

A fusion of Psb32 from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePsb32) with superfolder GFP was created for enhanced solubility and improved detection and purification. The fusion protein readily formed large hexagonal crystals belonging to space group P6₁22. A full data set extending to 2.3 Å resolution was collected at the Swiss Light Source. The phase problem could be solved by using only the sfGFP fusion partner or by using GFP and AtTLP18.3 from Arabidopsis thaliana as search models. Based on this expression construct, a versatile library of 24 vectors combining four different superfolder GFP variants and three affinity tags was generated to facilitate expression and screening of fluorescent fusion proteins.

Small Molecules Antagonise the MIA-Fibronectin Interaction in Malignant Melanoma.

Melanoma inhibitory activity (MIA), an extracellular protein highly expressed by malignant melanoma cells, plays an important functional role in melanoma development, progression, and metastasis. After its secretion, MIA directly interacts with extracellular matrix proteins, such as fibronectin (FN). By this mechanism, MIA actively facilitates focal cell detachment from surrounding structures and strongly promotes tumour cell invasion and migration. Hence, the molecular understanding of MIA’s function provides a promising target for the development of new strategies in malignant melanoma therapy. Here, we describe for the first time the discovery of small molecules that are able to disrupt the MIA-FN complex by selectively binding to a new druggable pocket, which we could identify on MIA by structural analysis and fragment-based screening. Our findings may inspire novel drug discovery efforts aiming at a therapeutically effective treatment of melanoma by targeting MIA.