Allosteric conformational barcodes direct signaling in the cell

Abstract

The cellular network is highly interconnected. Pathways merge and diverge. They proceed through shared proteins and may change directions. How are cellular pathways controlled and their directions decided, coded, and read? These questions become particularly acute when we consider that a small number of pathways, such as signaling pathways that regulate cell fates, cell proliferation, and cell death in development, are extensively exploited. This review focuses on these signaling questions from the structural standpoint and discusses the literature in this light. All co-occurring allosteric events (including posttranslational modifications, pathogen binding, and gain-of-function mutations) collectively tag the protein functional site with a unique barcode. The barcode shape is read by an interacting molecule, which transmits the signal. A conformational barcode provides an intracellular address label, which selectively favors binding to one partner and quenches binding to others, and, in this way, determines the pathway direction, and, eventually, the cell's response and fate.

Figure 1.. Posttranslational Phosphorylation of p53 Takes Place at Numerous Serine and Threonine Residues, Providing Barcodes for p53 Signaling

(A) Phosphorylation events in the N-terminal region mostly stabilize the p53 and lead to apoptosis. Several mutations in the core DNA-binding domain can (allosterically, by shifting the equilibrium) destabilize p53, leading to COP9 degradation. Phosphorylation in the C-terminal region can increase p53 activity and p53 DNA-binding affinity. “Signalome” is the cell-signaling network, consisting of highly connected functional modules (Ma’ayan et al., 2005). The COP9 signalosome (CSN) is a conserved protein complex that functions in the ubiquitin-proteasome pathway. It is a multisubunit protease that regulates the activity of cullin-RING ligase. It removes the post-translational modification of ubiquitin-like protein, Nedd8/Rub1, from thecullin component of the ubiquitin E3 ligase (i.e., deneddylation). In addition, it is associated with deubiquitination activity and protein kinase activities capable of phosphorylating important signaling regulators. CSN is a key player in the DNA-damage response, cell-cycle control, and gene expression (Wei and Deng, 2003; Wei et al., 2008). The figure illustrates that PTMs (here, phosphorylation) at the functional sites (i.e., orthosteric PTMs in the body of the text) can serve as barcodes. The N-terminal phosphorylation events are allosteric. In this paper, we focus on allosteric barcodes. (B) p53 is one of the most connected nodes in either the protein-protein interaction network or the gene regulation network. The central circle represents both wild-type p53 and isoforms; the light brown circles are phosphorylation events required for turning on/off specific p53-protein interactions. The blue circles represent proteins interacting with p53. Note that the phosphorylation of Ser15 and Ser20 blocks the p53-MDM2 interaction. The figure is adapted with permission from Tsai et al., (2009b).

Figure 2.. The Concept of a Conformational Barcode

A barcode is a readable representation of data relating to the object to which it is attached. Linear, or 1D, barcodes represent data by varying the widths of parallel lines and the spaces between them; geometric patterns in 2D can employ rectangles and various combinations, including hexagons. Protein-binding site barcodes are high-dimensional. (A) illustrates two acetylation marks (double orthosteric PTMs) on a histone H4 tail (red) recognized by the bromodomain (BD1) of Brdt (green), a testis-specific member of the BET protein family (Protein Data Bank [PDB] 2WP2; Morinière et al., 2009). That BD1 fails to bind monoacetylated H4 tail illustrates the essential role of combinatorial PTM barcode in interaction specificity. The acetylated lysines at positions 5 and 8 are shown. (B) highlights the role of a barcode in the cellular network. Proteins are depicted as nodes and interactions as edges. Some proteins (hubs) can have a large number of partners. A shared binding site of a hub protein can bind multiple partners. Proteins with similar color edges (purple or green) share the same site. Binding of a specific protein determines the signaling pathway. The binding event that takes place at a given time is largely governed by the barcode at the shared binding site.

Figure 3.. Schematic Illustrations of Binding by “Lock and Key,” “Induced Fit,” and “Conformational Selection” Models

(A) In (A), the drawing emphasizes the fact that the lock and key model only allows a binding to happen with an exact fit between complementary geometric shapes of ligand and receptor. The cross bar drawn in between implies no ligand-receptor binding can occur. Either the case of a receptor selecting among different ligands or the reverse of a ligand selecting among different receptors are depicted, with the binding exclusively determined by the exact lock-and-key fit criterion. (B and C) Different colors imply different receptors. In contrast to the lock and key model, the “induced fit” model highlighted in (B) indicates that there is no exact fit in the prebinding conformation between the ligand and receptor, as shown by the cross bar on the receptor fitted exactly to the ligand, and the consequent binding ends up with the receptor being induced to change the shape to fit into that of ligand. In the “conformational selection” model, the emphasis of the illustration in (C) is placed on the availability of pre-existing, different receptor conformations, and the ligand selects and binds the receptor via the lock and key criterion. Here, different conformations of the same receptor are in the same color (green). The figure emphasizes that the lock-and-key hypothesis considers many different receptor molecules. Each receptor has one specific structure. One of the receptor molecules (lock) fits the ligand (key). In contrast, in conformational selection, the ensemble consists of many distinct conformations of the same receptor molecule.

Figure 4.. Schematic Diagrams of Allosteric PTM Examples

(A) illustratesan activethyroid hormone-activating type 2 deiodinasewith a bound T4 substrate. It istransiently inactivated by ubiquitylation at the dimer interface. The allosteric action of the ubiquitylations is illustrated by the interacting blue ellipsoids, which propagatethe steric hindrance down to the T4 binding site (Sagar et al., 2007). (B) Progressive phosphorylation events up to a certain threshold number at the N-terminal region of circadian clock protein, FRQ, allosterically expose its protected middle region (purple circle) and lead to its own degradation (Querfurth et al., 2011). The allosteric action is illustrated by the interacting blue ellipsoids, which propagate the created electrostatic repulsion between the N-terminal and C-terminal of FRQ down to the exposed middle region (orange circle). (C-E) A phosphorylation-dependent activation of c-Cbl, a RING ubiquitin ligase that attenuate receptortyrosine kinase signal transduction, is illustrated by four captured crystal structures with a combination of two involved allosteric actions. (C) provides a model for the binding of substrate sequences (the ZAP-70 peptide) to the N-terminal SH2-containing tyrosine kinase-binding domain (TKBD) and of ubiquitin-conjugating enzyme (E2) and Tyr371 phosphorylation, together leading toward c-Cbl activation (Dou et al., 2012). In the absence of E2 and the TKBD substrate, the c-CBl (a single-subunit RING E3 that negatively regulates proteins by promoting ubiquitination and subsequent degradation; PDB 2Y1M) adopts a closed conformation, in which the E2-binding surface of RING subdomain associates with the TKBD. The allosteric effect of the ZAP-70 peptide binding partially opens the RING subdomain, making it favorable for E2 binding. This is clearly seen from the superposition of the two crystal structures (PDB 2Y1M and 2Y1N) shown in (D), which indicate the partial RING opening (the RING movement from red to green) due to the TKBD substrate binding (yellow ribbon). The subsequent E2 binding causes the RING subdomain to adopt an open conformation. Tyr371, which mediates the linker helix region (LHR) subdomain binding to the TKBD, secures the c-Cbl in an inactive state. The phosphorylation ofTyr371 activates c-Cbl by releasing LHR from TKBD, which then undergoes a large conformational change that brings the RING subdomain and E2 into proximity of the substrate. The large conformational change due to the pTyr371 is seen from the superposition of two crystal structures (PDB 1FBV and 4A4C) in (E). Both the side chains of Tyr371 (red) and pTyr371 (green) are given in space-fill models. (A) and (B) are adapted with permission from Nussinov et al., (2012)

Figure 5.. An Illustration of Distinct Molecular Switches that Can Be Decided bythe Diverse Allosteric Barcodes in Cytoplasmic Tyrosine Kinases

(A) These barcodes are expressed by three catalytic regulation mechanisms. These include (1) orthosteric phosphorylation of Tyr residues in the activation loop; (2) allosteric activation through binding at the N-lobe of kinase core domain; and (3) autoinhibitory binding at various allosteric sites. The first two regulation events are revealed in (A) with the example of active Fes kinase conformation (PDB 3CD3). The active site of Fes kinase is shown in the cleft between N-lobe (green) and C-lobe (yellow) subdomains, with the αC-helix positioned by the SH2 (red) subdomain in an active orientation as well as the activation loop, which is organized by the orthosteric phosphorylation of pY713to an active configuration. The allosteric intramolecular interactions are highlighted by the tight electrostatic interactions between residues Glu (E469 and E472) in SH2 and Arg (R609) in αC-helix. (B and C) The third autoinhibitory regulation event is depicted in (B) and (C) with the examples of Srcand Syk kinase, respectively. In (B), the inactive Src kinase (PDB 1FMK) is superimposed onto an active kinase (PDB 1YI6) with an orthosteric phosphorylation (pTyr416). When compared to the (superimposed) active kinase (orange), the disoriented N-lobe (yellow) from the inactive Src kinase reveals that the autoinhibitory mechanism is through the intramolecular SH3 (red) binding to the kinase N-lobe, mediated by the SH2-kinase linker (white). The autoinhibitory mechanism is also helped by the SH2 (green) binding to the kinase C-terminal through the phosphorylated Tyr (pTyr527). In (C), the inactive Syk kinase Zap-70 (PDB 2OZO) is superimposed on an active kinase (PDB 1U59) with dual orthosteric phosphorylation events (Y492 and Y493) and by an ITAM peptide (PDB 2OQ1) bound to both SH2 domains. The autoinhibitory mechanism suggested that the rigidification of SH2-kinase linker (blue) is due to the interaction with inter-SH2 linker (green), which reduces kinase flexibility at the catalytic cleft. Therefore, the activation of Syk kinase can be achieved either through the phosphorylation within SH2-kinase linker (pY315 and pY319) or binding of phosphorylated ITAM peptide (light green) with the two highlighted phosphorylated Tyr to the tandem SH2 subdomains. The mechanism of the disrupting interactions between inter-SH2 linker and SH2-kinase linker in the former action is orthosteric and is allosteric in the latter. See text for further detailed description of distinct molecular switches observed in cytoplasmic tyrosine kinases.