X-ray structure determination of human profilin II: A comparative structural analysis of human profilins

Human profilins are multifunctional, single-domain proteins which directly link the actin microfilament system to a variety of signalling pathways via two spatially distinct binding sites. Profilin binds to monomeric actin in a 1:1 complex, catalyzes the exchange of the actin-bound nucleotide and regulates actin filament barbed end assembly. Like SH3 domains, profilin has a surface-exposed aromatic patch which binds to proline-rich peptides. Various multidomain proteins including members of the Ena/VASP and formin families localize profilin:actin complexes through profilin:poly-L-proline interactions to particular cytoskeletal locations (e.g. focal adhesions, cleavage furrows). Humans express a basic (I) and an acidic (II) isoform of profilin which exhibit different affinities for peptides and proteins rich in proline residues. Here, we report the crystallization and X-ray structure determination of human profilin II to 2.2 A. This structure reveals an aromatic extension of the previously defined poly-L-proline binding site for profilin I. In contrast to serine 29 of profilin I, tyrosine 29 in profilin II is capable of forming an additional stacking interaction and a hydrogen bond with poly-L-proline which may account for the increased affinity of the second isoform for proline-rich peptides. Differential isoform specificity for proline-rich proteins may be attributed to the differences in charged and hydrophobic residues in and proximal to the poly-L-proline binding site. The actin-binding face remains nearly identical with the exception of five amino acid differences. These observations are important for the understanding of the functional and structural differences between these two classes of profilin isoforms.     

Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains

We have developed a class of binding proteins, called avimers, to overcome the limitations of antibodies and other immunoglobulin-based therapeutic proteins. Avimers are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display, generating multidomain proteins with binding and inhibitory properties. Linking multiple independent binding domains creates avidity and results in improved affinity and specificity compared with conventional single-epitope binding proteins. Other potential advantages over immunoglobulin domains include simple and efficient production of multitarget-specific molecules in Escherichia coli, improved thermostability and resistance to proteases. Avimers with sub-nM affinities were obtained against five targets. An avimer that inhibits interleukin 6 with 0.8 pM IC50 in cell-based assays is biologically active in two animal models.     

Structural plasticity underpins promiscuous binding of the prosurvival protein A1

Apoptotic pathways are regulated by protein-protein interactions. Interaction of the BH3 domains of proapoptotic Bcl-2 family proteins with the hydrophobic groove of prosurvival proteins is critical. Whereas some BH3 domains bind in a promiscuous manner, others exhibit considerable selectivity and the sequence characteristics that distinguish these activities are unclear. In this study, crystal structures of complexes between the prosurvival protein A1 and the BH3 domains from Puma, Bmf, Bak, and Bid have been solved. The structure of A1 is similar to that of other prosurvival proteins, although features, such as an acidic patch in the binding groove, may allow specific therapeutic modulation of apoptosis. Significant conformational plasticity was observed in the intermolecular interactions and these differences explain some of the variation in affinity. This study, in combination with published data, suggests that interactions between conserved residues demarcate optimal binding.     

Molecular recognition in bone morphogenetic protein (BMP)/receptor interaction

Bone morphogenetic proteins (BMPs) and other members of the TGF-beta superfamily are secreted signalling proteins determining the development, maintenance and regeneration of tissues and organs. These dimeric proteins bind, via multiple epitopes, two types of signalling receptor chains and numerous extracellular modulator proteins that stringently control their activity. Crystal structures of free ligands and of complexes with type I and type II receptor extracellular domains and with the modulator protein Noggin reveal structural epitopes that determine the affinity and specificity of the interactions. Modelling of a ternary complex BMP/(BMPR-IA(EC))2 / (ActR-II(EC))2 suggests a mechanism of receptor activation that does not rely on direct contacts between extracellular domains of the receptors. Mutational and interaction analyses indicate that the large hydrophobic core of the interface of BMP-2 (wrist epitope) with the type I receptor does not provide a hydrophobic hot spot for binding. Instead, main chain amide and carbonyl groups that are completely buried in the contact region represent major binding determinants. The affinity between ligand and receptor chains is probably strongly increased by two-fold interactions of the dimeric ligand and receptor chains that exist as homodimers in the membrane (avidity effects). BMP muteins with disrupted epitopes for receptor chains or modulator proteins provide clues for drug design and development.     

Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3

High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins.     

Chromatography of plasma proteins on immobilized Cibacron Blue F3-GA. Mechanism of the molecular interaction.

Fractionation of plasma proteins on immobilized Cibacron Blue F3-GA (Affi-gel Blue) under different conditions of pH, ionic strength and temperature was studied. At acidic pH the unbound proteins were eluted in order of increasing pI (the Affi-gel Blue behaving as ion-exchanger); at basic pH and at low ionic strength they were eluted in order of decreasing molecular weight (separation by diffusion-exclusion). For the proteins that were either retarded in comparison with substances of similar molecular characteristics, or that were bound to the resin, pseudo-ligand affinity or hydrophobic interactions were also implicated.     

Intercalating residues determine the mode of HMG1 domains A and B binding to cisplatin-modified DNA

Cisplatin exerts its anticancer activity by forming covalent adducts with DNA. High-mobility group (HMG)-domain proteins recognize the major 1,2-intrastrand cisplatin-DNA cross-links and can mediate cisplatin cytotoxicity. The crystal structure of HMG1 domain A bound to cisplatin-modified DNA, further analyzed here, reveals intercalation of a key Phe37 residue. Other published structures of HMG domains bound to DNA, including NHP6A and HMG-D, similarly indicate amino acid side chains intercalating into linear DNA to form a bend. To delineate the importance of such side chain intercalations and further to explore the binding modes of different HMG domains toward prebent DNA structures, site-directed mutagenesis was used to generate HMG1 domain A and domain B mutants. The affinities of these mutant proteins for cisplatin-modified DNA were determined in gel electrophoresis mobility shift assays. The results indicate that intercalating residues at positions 16 or 37 can both contribute to the binding affinity. The data further reveal that the length of the loop between helices I and II is not critical for binding affinity. Footprinting analyses indicate that the position of the intercalating residue dictates the binding mode of the domain toward platinated DNA. Both congruent and offset positioning of the HMG domain with respect to the locus of the cisplatin-induced bend in the DNA were encountered. Packing interactions in the crystal structure suggest how full-length HMG1 might bind to DNA by contacting more than one duplex simultaneously. Taken together, these results demonstrate that cisplatin modification of DNA provides an energetically favorable, prebent target for HMG domains, which bind to these targets through one or more side chain and favorable hydrophobic surface interactions.     

Hydrophobic matching of HIV-1 Vpu transmembrane helix-helix interactions is optimized for subcellular location

The HIV-1 accessory protein Vpu mediates the downregulation of several host cell proteins, an activity that is critical for viral replication in vivo. As the first step in directing cell-surface proteins to internal cellular compartments, and in many cases degradation, Vpu binds a subset of its target proteins through their transmembrane domains. Each of the known targets of Vpu are synthesized in the ER, and must traverse the different membrane environments found along the secretory pathway, thus it is important to consider how membrane composition might influence the interactions between Vpu and its targets. We have used Förster resonance energy transfer (FRET) to measure the oligomerization of Vpu with the transmembrane domains of target proteins in model membranes of varying lipid composition. Our data show that both lipid bilayer thickness and acyl chain order can significantly influence monomer-oligomer equilibria within the Vpu-target system. Changes in oligomerization levels were found to be non-specific with no single Vpu-target interaction being favored under any condition. Our analysis of the influence of the membrane environment on the strength of helix-helix interactions between Vpu and its targets in vitro suggests that the strength of Vpu-target interactions in vivo will be partially dependent on the membrane environment found in specific membrane compartments.