Anne Poupon

DECIPHERING PROTEIN SEQUENCE INFORMATION THROUGH HYDROPHOBIC CLUSTER ANALYSIS (HCA): CURRENT STATUS AND PERSPECTIVES

Abstract. Ten years after the idea of hydrophobic cluster analysis (HCA) was conceived and first pub lished, theoretical and practical experience has shown this unconventional method of protein sequence anal ysis to be particularly efficient and sensitive, especially with families of sequences sharing low levels of se quence identity. This extreme sensitivity has made it possible to predict the functions of genes whose se quence similarities are hardly if at all detectable by current one-dimensional (1D) methods alone, and of fers a new way to explore the enormous amount of data generated by genome sequencing. HCA also pro vides original tools to understand fundamental fea tures of protein stability and folding. Since the last review of HCA published in 1990 [1], significant im provements have been made and several new facets have been addressed. Here we wish to update and summarize this information.

Community-wide assessment of protein-interface modeling suggests improvements to design methodology

The CAPRI (Critical Assessment of Predicted Interactions) and CASP (Critical Assessment of protein Structure Prediction) experiments have demonstrated the power of community-wide tests of methodology in assessing the current state of the art and spurring progress in the very challenging areas of protein docking and structure prediction. We sought to bring the power of community-wide experiments to bear on a very challenging protein design problem that provides a complementary but equally fundamental test of current understanding of protein-binding thermodynamics. We have generated a number of designed protein-protein interfaces with very favorable computed binding energies but which do not appear to be formed in experiments, suggesting that there may be important physical chemistry missing in the energy calculations. A total of 28 research groups took up the challenge of determining what is missing: we provided structures of 87 designed complexes and 120 naturally occurring complexes and asked participants to identify energetic contributions and/or structural features that distinguish between the two sets. The community found that electrostatics and solvation terms partially distinguish the designs from the natural complexes, largely due to the nonpolar character of the designed interactions. Beyond this polarity difference, the community found that the designed binding surfaces were, on average, structurally less embedded in the designed monomers, suggesting that backbone conformational rigidity at the designed surface is important for realization of the designed function. These results can be used to improve computational design strategies, but there is still much to be learned; for example, one designed complex, which does form in experiments, was classified by all metrics as a nonbinder.

Structure of Protein Phosphatase Methyltransferase 1 (PPM1), a Leucine Carboxyl Methyltransferase Involved in the Regulation of Protein Phosphatase 2A Activity

The important role of the serine/threonine protein phosphatase 2A (PP2A) in various cellular processes requires a precise and dynamic regulation of PP2A activity, localization, and substrate specificity. The regulation of the function of PP2A involves the reversible methylation of the COOH group of the C-terminal leucine of the catalytic subunit, which, in turn, controls the enzymes heteromultimeric composition and confers different protein recognition and substrate specificity. We have determined the structure of PPM1, the yeast methyltransferase responsible for methylation of PP2A. The structure of PPM1 reveals a common S-adenosyl-l-methionine-dependent methyltransferase fold, with several insertions conferring the specific function and substrate recognition. The complexes with the S-adenosyl-l-methionine methyl donor and the S-adenosyl-l-homocysteine product and inhibitor unambiguously revealed the co-substrate binding site and provided a convincing hypothesis for the PP2A C-terminal peptide binding site. The structure of PPM1 in a second crystal form provides clues to the dynamic nature of the PPM1/PP2A interaction.

POPULATIONS OF HYDROPHOBIC AMINO ACIDS WITHIN PROTEIN GLOBULAR DOMAINS : IDENTIFICATION OF CONSERVED TOPOHYDROPHOBIC POSITIONS

The 3D structural comparison of families of divergent homologous domains revealed two main populations of hydrophobic amino acids, one with a low and the other with a significantly higher mean solvent accessibility, allowing two regions of the core of protein globular domains to be distinguished. The side chains of hydrophobic amino acids in topologically conserved positions (positions in the structural alignment where only hydrophobic amino acids are found), which we call topohydrophobic positions, are considerably less dispersed than those of the other amino acids (hydrophobic or not). Mean distances between gravity centers of amino acids in topohydrophobic positions are significantly shorter than those for non‐topohydrophobic positions and show that the corresponding amino acids are almost all in direct contact in the inner core of globular domains. This study also showed that the small number of topohydrophobic positions is a characteristic of the structural differences between proteins of a family. This criterion is independent of the sequence identity between the sequences and of the root‐mean‐square distance between their corresponding structures. Using sensitive sequence alignment processes it will be possible, for many protein families, to identify topohydrophobic positions from sequences only. Proteins 33:329–342, 1998.

A new protein--protein docking scoring function based on interface residue properties

MOTIVATION Protein-protein complexes are known to play key roles in many cellular processes. However, they are often not accessible to experimental study because of their low stability and difficulty to produce the proteins and assemble them in native conformation. Thus, docking algorithms have been developed to provide an in silico approach of the problem. A protein-protein docking procedure traditionally consists of two successive tasks: a search algorithm generates a large number of candidate solutions, and then a scoring function is used to rank them. RESULTS To address the second step, we developed a scoring function based on a Voronoï tessellation of the protein three-dimensional structure. We showed that the Voronoï representation may be used to describe in a simplified but useful manner, the geometric and physico-chemical complementarities of two molecular surfaces. We measured a set of parameters on native protein-protein complexes and on decoys, and used them as attributes in several statistical learning procedures: a logistic function, Support Vector Machines (SVM), and a genetic algorithm. For the later, we used ROGER, a genetic algorithm designed to optimize the area under the receiver operating characteristics curve. To further test the scores derived with ROGER, we ranked models generated by two different docking algorithms on targets of a blind prediction experiment, improving in almost all cases the rank of native-like solutions. AVAILABILITY http://genomics.eu.org/spip/-Bioinformatics-tools-

Mapping the follicle-stimulating hormone-induced signaling networks

Follicle-stimulating hormone (FSH) is a central regulator of male and female reproductive function. Over the last decade, there has been a growing perception of the complexity associated with FSH-induced cellular signaling. It is now clear that the canonical Gs/cAMP/PKA pathway is not the sole mechanism that must be considered in FSH biological actions. In parallel, consistent with the emerging concept of biased agonism, several examples of ligand-mediated selective signaling pathway activation by gonadotropin receptors have been reported. In this context, it is important to gain an integrative view of the signaling pathways induced by FSH and how they interconnect to form a network. In this review, we propose a first attempt at building topological maps of various pathways known to be involved in the FSH-induced signaling network. We discuss the multiple facets of FSH-induced signaling and how they converge to the hormone integrated biological response. Despite of their incompleteness, these maps of the FSH-induced signaling network represent a first step toward gaining a system-level comprehension of this hormone’s actions, which may ultimately facilitate the discovery of novel regulatory processes and therapeutic strategies for infertility and non-steroidal contraception.

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.

Predicting the protein folding nucleus from a sequence

Understanding the mechanism of protein folding would allow prediction of the three‐dimensional structure from sequence data alone. It has been shown that small proteins fold in a small number of kinetic steps and that significantly populated intermediate states exist for some of them. Studies of these intermediates have demonstrated the existence of specific interactions established during the initial stages of folding. Comparison of the amino acids participating in these specific and essential interactions and constituting the folding nucleus with conserved hydrophobic positions of a given fold shows a striking correspondence. This finding opens the perspective of predicting the folding nucleus knowing only a set of divergent sequences of a protein family.

Competing G protein-coupled receptor kinases balance G protein and β-arrestin signaling

Seven‐transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β‐arrestins, whose recruitment to the activated receptor is regulated by G protein‐coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal‐regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT1AR) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)‐based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well‐established function in the desensitization of G‐protein activation, GRK2 exerts a strong negative effect on β‐arrestin‐dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2‐dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT1AR, and HEK293 cells expressing other 7TMRs.

Sequence and structural features of the T-fold, an original tunnelling building unit.

A similar fold has been found in four archetype enzymes that perform different functions. This new fold has been named the T‐fold because it is found in multimeric proteins crossed by a tunnel. The T‐fold consists of an antiparallel β‐sheet of four sequential strands, and two antiparallel helices between the second and third strand, layered on the concave side of the β‐sheet. The presently known T‐fold proteins share a high structural similarity (a mean of 1.4 Å root mean square (r.m.s.) deviation on the common core) while they only exhibit a low level of sequence identity (a mean of 10.5% on the aligned regions). They bind to substrates belonging to the purine or pterin families, and share a fold‐related binding site with a glutamate or glutamine residue anchoring the substrate and a lot of conserved interactions. They also share a similar oligomerization mode: several T‐folds join together to form a β2nαn barrel, then two barrels join together in a head‐to‐head fashion to made up the native enzymes. The T‐fold has the characteristics of a globular domain, with a hydrophobic core and a clearly defined topohydrophobic network. It defines a new class of common folds or recurrent domains found in distantly related proteins. However, it is likely not stable in monomeric form and until now is only observed in association with other T‐folds through multimerization. Proteins 2000;39:142–154.