Philippe Minard

Crystal structure of the yeast phox homology (PX) domain protein Grd19p complexed to phosphatidylinositol-3-phosphate

Phox homology (PX) domains have been recently identified in a number of different proteins and are involved in various cellular functions such as vacuolar targeting and membrane protein trafficking. It was shown that these modules of about 130 amino acids specifically binding to phosphoinositides and that this interaction is crucial for their cellular function. The yeast genome contains 17 PX domain proteins. One of these, Grd19p, is involved in the localization of the late Golgi membrane proteins DPAP A and Kex2p. Grd19p consists of the PX domain with 30 extra residues at the N-terminal and is homologous to the functionally characterized human sorting nexin protein SNX3. We determined the 2.0 Å crystal structure of Grd19p in the free form and in complex with d-myo-phosphatidylinositol 3-phosphate (diC4PtdIns(3)P), representing the first case of both free and ligand-bound conformations of the same PX module. The ligand occupies a well defined positively charged binding pocket at the interface between the β-sheet and α-helical parts of the molecule. The structure of the free and bound protein are globally similar but show some significant differences in a region containing a polyproline peptide and a putative membrane attachment site.

Refolding strategies from inclusion bodies in a structural genomics project

AbstractThe South-Paris Yeast Structural Genomics Project aims at systematically expressing, purifying and determining the structure of S. cerevisiae proteins with no detectable homology to proteins of known structure (http://genomics.eu.org/). We brought 250 yeast ORFs to expression in E. coli, but 37% of them form inclusion bodies. This important fraction of proteins that are well expressed but lost for structural studies prompted us to test methodologies to recover these proteins. Three different strategies were explored in parallel on a set of 20 proteins: (1) refolding from solubilized inclusion bodies using an original and fast 96-well plates screening test, (2) co-expression of the targets in E. coli with DnaK-DnaJ-GrpE and GroEL-GroES chaperones, and (3) use of the cell-free expression system. Most of the tested proteins (17/20) could be resolubilized at least by one approach, but the subsequent purification proved to be difficult for most of them. abbreviations: GdnHCl – guanidine hydrochloride; IPTG – isopropyl-β-d-thiogalactopyranoside; NMR – nuclear magnetic resonance spectroscopy; ORF – open reading frame; PCR – polymerase chain reaction; SDS-PAGE – sodium dodecylsulfate-polyacrylamide gel electrophoresis; TCA – trichloroacetic acid; β-SH – 2-mercaptoethanol.

Artificial proteins from combinatorial approaches

How do we create new artificial proteins? In this review, we present a range of experimental approaches based on combinatorial and directed evolution methods used to explore sequence space and recreate structured or active proteins. These approaches can help to understand constraints of natural evolution and can lead to new useful proteins. Strategies such as binary patterning or modular assembly can efficiently speed structural and functional innovation. Many natural protein architectures are symmetric or repeated and presumably have emerged by coalescence of simpler fragments. This process can be experimentally reproduced; a range of artificial proteins obtained from idealized fragments has recently been described and some of these have already found direct applications.

Amphipol-mediated screening of molecular orthoses specific for membrane protein targets.

Specific, tight-binding protein partners are valuable helpers to facilitate membrane protein (MP) crystallization, because they can i) stabilize the protein, ii) reduce its conformational heterogeneity, and iii) increase the polar surface from which well-ordered crystals can grow. The design and production of a new family of synthetic scaffolds (dubbed αReps, for “artificial alpha repeat protein”) have been recently described. The stabilization and immobilization of MPs in a functional state are an absolute prerequisite for the screening of binders that recognize specifically their native conformation. We present here a general procedure for the selection of αReps specific of any MP. It relies on the use of biotinylated amphipols, which act as a universal “Velcro” to stabilize, and immobilize MP targets onto streptavidin-coated solid supports, thus doing away with the need to tag the protein itself.

Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)

We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a “filtration” procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×109 independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties.

A structural genomics initiative on yeast proteins.

A canonical structural genomics programme is being conducted at the Paris-Sud campus area on bakers yeast proteins. Experimental strategies, first results and identified bottlenecks are presented. The actual or potential contributions to the structural genomics of several experimental structure-determination methods are discussed.

Specific GFP-binding artificial proteins (αRep): a new tool for in vitro to live cell applications

Artificial proteins, named αRep, binding tightly and specifically to EGFP are described. The structures of αRep–EGFP complexes explain how αRep recognize their cognate partner. Specific αRep can be used for biochemical or live cells experiments.

Crystal Structure of the Ygr205W Protein from Saccharomyces Cerevisiae: Close Structural Resemblance to E.Coli Pantothenate Kinase

The protein product of the YGR205w gene of Saccharomyces cerevisiae was targeted as part of our yeast structural genomics project. YGR205w codes for a small (290 amino acids) protein with unknown structure and function. The only recognizable sequence feature is the presence of a Walker A motif (P loop) indicating a possible nucleotide binding/converting function. We determined the three‐dimensional crystal structure of Se‐methionine substituted protein using multiple anomalous diffraction. The structure revealed a well known mononucleotide fold and strong resemblance to the structure of small metabolite phosphorylating enzymes such as pantothenate and phosphoribulo kinase. Biochemical experiments show that YGR205w binds specifically ATP and, less tightly, ADP. The structure also revealed the presence of two bound sulphate ions, occupying opposite niches in a canyon that corresponds to the active site of the protein. One sulphate is bound to the P‐loop in a position that corresponds to the position of β‐phosphate in mononucleotide protein ATP complex, suggesting the protein is indeed a kinase. The nature of the phosphate accepting substrate remains to be determined. Proteins 2004;54:000–000.

Disulfide Bond Substitution by Directed Evolution in an Engineered Binding Protein

Breaking ties: The antitumour protein, neocarzinostatin (NCS), is one of the few drug‐carrying proteins used in human therapeutics. However, the presence of disulfide bonds limits this proteins potential development for many applications. This study describes a generic directed‐evolution approach starting from NCS‐3.24 (shown in the figure complexed with two testosterone molecules) to engineer stable disulfide‐free NCS variants suitable for a variety of purposes, including intracellular applications.

Structural and functional analysis of the fibronectin-binding protein FNE from Streptococcus equi spp. equi

Streptococcus equi is a horse pathogen belonging to Lancefield group C. Infection by S. equi ssp. equi causes strangles, a serious and highly contagious disease of the upper respiratory tract. S. equi ssp. equi secretes a fibronectin (Fn)‐binding protein, FNE, that does not contain cell wall‐anchoring motifs. FNE binds to the gelatin‐binding domain (GBD) of Fn, composed of the motifs 6FI12FII789FI. FNE lacks the canonical Fn‐binding peptide repeats observed in many microbial surface components recognizing adhesive matrix molecules. We found that the interaction between FNE and the human GBD is mediated by the binding of the disordered C‐terminal region (residues 208–262) of FNE to the 789FI GBD subfragment. The crystal structure of FNE showed that it is similar to the minor pilus protein Spy0125 of Streptococcus pyogenes, found at the end of pilus polymers and responsible for adhesion. FNE and Spy0125 both have a superimposable internal thioester bond between highly conserved Cys and Gln residues. Small‐angle X‐ray scattering of the FNE–789FI complex provided a model that aligns the C‐terminal peptide of FNE with the E‐strands of the FI domains, adopting the β‐zipper extension model observed in previous structures of microbial surface components recognizing adhesive matrix molecule adhesion peptides bound to FI domains.