Engineering hepatocyte growth factor fragments with high stability and activity as Met receptor agonists and antagonists

The Met receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) play an important role in mediating both tumor progression and tissue regeneration. The N-terminal and first Kringle domains (NK1) of HGF comprise a naturally occurring splice variant that retains the ability to activate the Met receptor. However, NK1 is a weak agonist and is relatively unstable, limiting its therapeutic potential. Here, we engineered NK1 mutants with improved biochemical and biophysical properties that function as Met receptor agonists or antagonists. We first engineered NK1 for increased stability and recombinant expression yield using directed evolution. The NK1 variants isolated from our library screens acted as weak Met receptor antagonists due to a mutation at the NK1 homodimerization interface. We introduced point mutations that restored this NK1 homodimerization interface to create an agonistic ligand, or that further disrupted this interface to create more effective antagonists. The rationally engineered antagonists exhibited melting temperatures up to approximately 64 °C, a 15 °C improvement over antagonists derived from wild-type NK1, and approximately 40-fold improvement in expression yield. Next, we created disulfide-linked NK1 homodimers through introduction of an N-terminal cysteine residue. These covalent dimers exhibited nearly an order of magnitude improved agonistic activity compared to wild-type NK1, approaching the activity of full-length HGF. Moreover, covalent NK1 dimers formed from agonistic or antagonistic monomeric subunits elicited similar activity, further signifying that NK1 dimerization mediates agonistic activity. These engineered NK1 proteins are promising candidates for therapeutic development and will be useful tools for further exploring determinants of Met receptor activation.     

Pharmacological modulation of chemokine receptor function

G protein-coupled chemokine receptors and their peptidergic ligands are interesting therapeutic targets due to their involvement in various immune-related diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, HIV-1 infection and cancer. To tackle these diseases, a lot of effort has been focused on discovery and development of small-molecule chemokine receptor antagonists. This has been rewarded by the market approval of two novel chemokine receptor inhibitors, AMD3100 (CXCR4) and Maraviroc (CCR5) for stem cell mobilization and treatment of HIV-1 infection respectively. The recent GPCR crystal structures together with mutagenesis and pharmacological studies have aided in understanding how small-molecule ligands interact with chemokine receptors. Many of these ligands display behaviour deviating from simple competition and do not interact with the chemokine binding site, providing evidence for an allosteric mode of action. This review aims to give an overview of the evidence supporting modulation of this intriguing receptor family by a range of ligands, including small molecules, peptides and antibodies. Moreover, the computer-assisted modelling of chemokine receptor-ligand interactions is discussed in view of GPCR crystal structures. Finally, the implications of concepts such as functional selectivity and chemokine receptor dimerization are considered.     

Modelling the interdependence between the stoichiometry of receptor oligomerization and ligand binding for a coexisting dimer/tetramer receptor system

Many G protein-coupled receptors have been shown to exist as oligomers, but the oligomerization state and the effects of this on receptor function are unclear. For some G protein-coupled receptors, in ligand binding assays, different radioligands provide different maximal binding capacities. Here we have developed mathematical models for co-expressed dimeric and tetrameric species of receptors. We have considered models where the dimers and tetramers are in equilibrium and where they do not interconvert and we have also considered the potential influence of the ligands on the degree of oligomerization. By analogy with agonist efficacy, we have considered ligands that promote, inhibit or have no effect on oligomerization. Cell surface receptor expression and the intrinsic capacity of receptors to oligomerize are quantitative parameters of the equations. The models can account for differences in the maximal binding capacities of radioligands in different preparations of receptors and provide a conceptual framework for simulation and data fitting in complex oligomeric receptor situations.     

Molecular architecture of the ErbB2 extracellular domain homodimer

Human epidermal growth factor receptors (HERs or ErbBs) play crucial roles in numerous cellular processes. ErbB2 is a key member of ErbB family, and its overexpression is recognized as a frequent molecular abnormality. In cancer, this overexpression correlates with aggressive disease and poor patient outcomes. Dimer-dependent phosphorylation is a key event for the signal transduction of ErbBs. However, the molecular mechanism of the dimerization of ErbB2 remains elusive. In the present work, we report the homodimer architecture of the ErbB2 extracellular domain (ECD) which is unique compared with other dimer-models of ErbBs. The structure of the ErbB2 ECD homodimer represents a “back to head” interaction, in which a protruding β-hairpin arm in domain II of one ErbB2 protomer is inserted into a C-shaped pocket created by domains I–III of the adjacent ErbB2 protomer. This dimerized architecture and its impact on the phosphorylation of ErbB2 intracellular domain were further verified by a mutagenesis study. We also elucidated the different impacts of two clinically administered therapeutic antibodies, trastuzumab and pertuzumab, on ErbB2 dimerization. This information not only provides an understanding of the molecular mechanism of ErbBs dimerization but also elucidates ErbB2-targeted therapy at the molecular level.     

Heteromeric Dopamine Receptor Signaling Complexes: Emerging Neurobiology and Disease Relevance

The pharmacological modification of dopamine transmission has long been employed as a therapeutic tool in the treatment of many mental health disorders. However, as many of the pharmacotherapies today are not without significant side effects, or they alleviate only a particular subset of symptoms, the identification of novel therapeutic targets is imperative. In light of these challenges, the recognition that dopamine receptors can form heteromers has significantly expanded the range of physiologically relevant signaling complexes as well as potential drug targets. Furthermore, as the physiology and disease relevance of these receptor heteromers is further understood, their ability to exhibit pharmacological and functional properties distinct from their constituent receptors, or modulate the function of endogenous homomeric receptor complexes, may allow for the development of alternate therapeutic strategies and provide new avenues for drug design. In this review, we describe the emerging neurobiology of the known dopamine receptor heteromers, their physiological relevance in brain, and discuss the potential role of these receptor complexes in neuropsychiatric disease. We highlight their value as targets for future drug development and discuss innovative research strategies designed to selectively target these dopamine receptor heteromers in the search for novel and clinically efficacious pharmacotherapies.     

Structural and energetic determinants of adhesive binding specificity in type I cadherins

Type I cadherin cell-adhesion proteins are similar in sequence and structure and yet are different enough to mediate highly specific cell–cell recognition phenomena. It has previously been shown that small differences in the homophilic and heterophilic binding affinities of different type I family members can account for the differential cell-sorting behavior. Here we use a combination of X-ray crystallography, analytical ultracentrifugation, surface plasmon resonance and double electron-electron resonance (DEER) electron paramagnetic resonance spectroscopy to identify the molecular determinants of type I cadherin dimerization affinities. Small changes in sequence are found to produce subtle structural and dynamical changes that impact relative affinities, in part via electrostatic and hydrophobic interactions, and in part through entropic effects because of increased conformational heterogeneity in the bound states as revealed by DEER distance mapping in the dimers. These findings highlight the remarkable ability of evolution to exploit a wide range of molecular properties to produce closely related members of the same protein family that have affinity differences finely tuned to mediate their biological roles.In metazoans, the elaboration and maintenance of multicellular architectures relies upon the ability of cells to specifically adhere to one another. Cadherins constitute a superfamily of single-pass transmembrane proteins that can confer such specific adhesive properties to cells (1). In particular, the classical type I and type II cadherins, which are only found in vertebrates and are characterized by an extracellular region comprised of five extracellular cadherin (EC) domains, have been shown to help drive cell-patterning behavior in numerous settings: for example, in morphogenesis (2–4) and in neural patterning (5, 6). Cells expressing the same classical cadherin on their surface generally aggregate through homophilic interactions, whereas cells expressing different cadherins segregate into distinct layers that, in at least some instances, remain in contact with each other through heterophilic binding (7–9).Cell adhesion by classic cadherins is mediated by the dimerization of cadherin extracellular domains emanating from apposed cell surfaces through an interface confined to the N-terminal EC1 domain (Fig. 1A). Numerous crystal structures have revealed the atomic details of the trans (i.e., between cells) dimerization interface for three type I cadherins: C-, E-, and N-cadherins (10–13). In all three cases, the dimer partner molecules swap their N-terminal β-strand (the A*-strand), whose conserved Trp2 residues provide an “anchor” for the adhesive interface by docking into a complementary hydrophobic pocket in the partner protomer (Fig. 1A). A second dimerization interface that can form in the trans orientation has been observed in crystal structures of mutants of both type I and type II classical cadherins. Specifically, numerous mutations that disrupt strand-swapping in E-cadherin result in the formation of a distinct, lower-affinity homodimer—called the X-dimer because of its appearance—with a binding interface localized around the Ca²⁺-binding interdomain linker region between EC1 and EC2 (14–16) (Fig. 1B). It has been demonstrated that for E-cadherin this interface functions as a kinetic intermediate in the formation of the strand-swapped dimer (16–18) (Fig. 1B).Open in a separate windowFig. 1.Trans-dimerization of type I classical cadherins. Protomers emerging from apposed cells are shown in blue and orange, and calcium ions are shown as green spheres. (A) Schematic illustration of type I classical cadherin trans dimer on cell membranes. Extracellular regions dimerize through an interface located in their EC1 domain in which the N-terminal β-strands are swapped, and Trp2 from each protomer is docked in its partner’s hydrophobic pocket (expanded view, PDB ID code 2QVF). The A* strand, which consists of the first three residues in the sequence and swaps during dimerization, and the A strand, which consists of the last four residues (7–10) in the first β-strand, are indicated. (B) Reaction scheme showing the X-dimer acting as a kinetic intermediate during the formation of the strand-swapped dimer, in E-cadherin. In the X-dimer (PDB ID code 1FF5), Trp2 is docked in its own protomer’s hydrophobic pocket. (C) Schematic illustration of positions of key mutations investigated in this work. They are present on both protomers, but for clarity are shown on one protomer only. The X-dimer–incompetent mutation is indicated in olive (Right); mutations that directly disrupt the strand-swap interface are indicated in pink, those that line the floor of the tryptophan pocket in teal, and those that affect the electrostatic potential at the swap interface are in dark blue (Left).There is a considerable body of evidence demonstrating that the adhesive properties of cells reflect the binding properties of the cadherin molecules they express, and that these properties depend critically on the strand-swapped interface (11, 19–22). Despite their homotypic cell-sorting behavior, biophysical studies with purified cadherin ectodomains have shown that cadherins bind both homophilically and heterophilically (9, 23, 24). For the case of E- and N-cadherins, which have slightly different homophilic dimerization free energies, −6.25 kcal/mol for N-cadherin and −5.47 kcal/mol for E-cadherin, the binding free energy associated with heterophilic N-/E-dimerization is intermediate between these two homophilic values (9). We showed that this combination of homophilic and heterophilic molecular binding affinities predict the observed cell sorting behavior of N- and E-cadherin–expressing cells (9). The link between molecular and cellular behavior depends on the formation of multiple dimers at the contact surface between two adhesive cells, which amplifies the small affinity differences at the level of single molecules (25). It thus appears that subtle differences in the sequence and structure of type I cadherins can have profound effects on cellular behavior, but the molecular origins of these differences have not yet been determined.Elucidating the source of about 1 kcal/mol difference in binding free energy between two very similar molecules poses a challenging problem. Strand-swapping makes the problem even more complicated because it is not possible to deduce the binding-affinity determinants from the crystal structures of the binding interfaces alone. This is because an important consequence of β-strand swapping, or more generally of 3D domain swapping, is that each interaction that stabilizes the dimeric conformation is also formed intramolecularly in the “closed monomer” conformation (where the N-terminal strand is bound by its own protomer rather than swapping with a partner molecule). As a result, to a first approximation, interactions formed in the dimer must be broken in the monomer so that the net dimerization free energy is a result of subtle differences between very similar energetic terms (25). We recently found that one source of dimerization energy difference between the swapped dimer and the monomer state of E-cadherin is conformational strain in the A*/A-strand (Fig. 1A) in the monomer that is not present in the dimer, thus favoring dimerization (26). This mechanism is likely relevant for N-cadherin as well (27).Here we report studies aimed at understanding the relationship between the sequences, structures, and dimerization free energies of type I classical cadherins. We report four new crystal structures of adhesive EC1–EC2 fragments: the P-cadherin swapped dimer, a mutant N-cadherin that reveals its X-dimer structure, and two affinity-mutants of N-cadherin. We use analytical ultracentrifugation (AUC) to quantify the homophilic binding affinities for each type I cadherin, surface plasmon resonance biosensor analysis (SPR) to characterize heterophilic binding between type I cadherin pairs, and double electron-electron resonance (DEER) electron paramagnetic resonance (EPR) experiments to characterize dimer interactions and dynamics in solution. The combined AUC and SPR measurements provide a nearly complete interaction matrix for this important family of cell-adhesion proteins, and the X-ray and DEER data make it possible to interpret the affinity measurements in structural and dynamical terms. Our study demonstrates how multiple biophysical and structural approaches can be used in concert to address mechanistic questions that are not answerable with a more limited repertoire of technologies. Importantly, this process clarifies design principles in the type I cadherin subfamily and, in addition, reveals remarkable examples of the fine-tuning of binding specificities for closely related proteins. In this regard, a particularly novel finding is that individual cadherins appear to exist as an equilibrium ensemble of multiple conformational states and that the entropic contribution of this dynamic behavior may have important effects on binding affinities and consequently on cell adhesive specificity.     

Electrochemical reduction of N-thioamidoimidates: application to the synthesis of thiazolo[5,4-d]thiazoles

The reduction of N-thioamidoimidates (1) has been examined in aprotic media at a mercury electrode. As shown by cyclic voltammetry at fast scan rates and controlled potential electrolysis, an overall irreversible one electron transfer is followed by a rapid second order chemical reaction leading to a dimer which involves to thiazolo[5,4-d]thiazole by intramolecular cyclization.