Reuse of structural domain–domain interactions in protein networks

Background  

Protein interactions are thought to be largely mediated by interactions between structural domains. Databases such as iPfam relate interactions in protein structures to known domain families. Here, we investigate how the domain interactions from the iPfam database are distributed in protein interactions taken from the HPRD, MPact, BioGRID, DIP and IntAct databases.     

The β1 domain of protein G can replace the chorismate mutase domain of the T-protein

T-protein is composed of chorismate mutase (AroQ(T)) fused to the N-terminus of prephenate dehydrogenase (TyrA). Here, we report the replacement of AroQ(T) with the β1-domain of protein G (Gβ1). The TyrA domain shows a strong dehydrogenase activity within the context of this fusion, and our data indicate that Gβ1-TyrA folds into a dimeric conformation. Amino acid substitutions in the Gβ1 domain of Gβ1-TyrA identified residues involved in stabilizing the TyrA dimeric conformation. Gβ1 substitutions in the N-terminal β-hairpin eliminated Gβ1-TyrA expression, whereas Gβ1-TyrA tolerated Gβ1 substitutions in the C-terminal β-hairpin and in the α-helix. All of the characterized variants folded into a dimeric conformation. The importance of the β2-strand in forming a Gβ1 homo-dimerization interface explains the relevance of the first-β-hairpin in stabilizing the dimeric TyrA protein.     

Understanding the role of domain–domain linkers in the spatial orientation of domains in multi-domain proteins

Inter-domain linkers (IDLs)’ bridge flanking domains and support inter-domain communication in multi-domain proteins. Their sequence and conformational preferences enable them to carry out varied functions. They also provide sufficient flexibility to facilitate domain motions and, in conjunction with the interacting interfaces, they also regulate the inter-domain geometry (IDG). In spite of the basic intuitive understanding of the inter-domain orientations with respect to linker conformations and interfaces, we still do not entirely understand the precise relationship among the three. We show that IDG is evolutionarily well conserved and is constrained by the domain–domain interface interactions. The IDLs modulate the interactions by varying their lengths, conformations and local structure, thereby affecting the overall IDG. Results of our analysis provide guidelines in modelling of multi-domain proteins from the tertiary structures of constituent domain components.     

Equilibrium unfolding of the PDZ domain of β2-syntrophin

β2-syntrophin, a dystrophin-associated protein, plays a pivotal role in insulin secretion by pancreatic β-cells. It contains a PDZ domain (β2S-PDZ) that, in complex with protein-tyrosine phosphatase ICA512, anchors the dense insulin granules to actin filaments. The phosphorylation state of β2-syntrophin allosterically regulates the affinity of β2S-PDZ for ICA512, and the disruption of the complex triggers the mobilization of the insulin granule stores. Here, we investigate the thermal unfolding of β2S-PDZ at different pH and urea concentrations. Our results indicate that, unlike other PDZ domains, β2S-PDZ is marginally stable. Thermal denaturation experiments show broad transitions and cold denaturation, and a two-state model fit reveals a significant unfolded fraction under physiological conditions. Furthermore, T(m) and T(max) denaturant-dependent shifts and noncoincidence of melting curves monitored at different wavelengths suggest that two-state and three-state models fail to explain the equilibrium data properly and are in better agreement with a downhill scenario. Its higher stability at pH >9 and the results of molecular dynamics simulations indicate that this behavior of β2S-PDZ might be related to its charge distribution. All together, our results suggest a link between the conformational plasticity of the native ensemble of this PDZ domain and the regulation of insulin secretion.     

Secretion of N-terminal domain of α-dystroglycan in cerebrospinal fluid

α-Dystroglycan (α-DG) plays crucial roles in maintaining the stability of cells. We demonstrated previously that the N-terminal domain of α-DG (α-DG-N) is secreted by cultured cells into the culture medium. In the present study, to clarify its function in vivo, we generated a monoclonal antibody against α-DG-N and investigated the secretion of α-DG-N in human cerebrospinal fluid (CSF). Interestingly, we found that a considerable amount of α-DG-N was present in CSF. α-DG-N in CSF was a sialylated glycoprotein with both N- and O-linked glycan. These observations suggest that secreted α-DG-N may be transported via CSF and have yet unidentified effects on the nervous system.     

Secondary structure of the C-terminal domain of the tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus: a novel type of anticodon binding domain?

The tyrosyl-tRNA synthetase catalyzes the activation of tyrosine and its coupling to the cognate tRNA. The enzyme is made of two domains: an N-terminal catalytic domain and a C-terminal domain that is necessary for tRNA binding and for which it was not possible to determine the structure by X-ray crystallography. We determined the secondary structure of the C-terminal domain of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus by nuclear magnetic resonance methods and found that it is of the alpha+beta type. Its arrangement differs from those of the other anticodon binding domains whose structure is known. We also found that the isolated C-terminal domain behaves as a folded globular protein, and we suggest the presence of a flexible linker between the two domains.     

Basis of the intrinsic flexibility of the Cε3 domain of IgE

Allergic reactions are triggered by the interaction between IgE and its high-affinity receptor, FcεRI. Various studies have mapped the interaction surface between IgE and its cellular receptors to the third constant domain of IgE (Cε3). The isolated Cε3 domain has been shown to exist as a molten globule, and the domain retains significant flexibility within the context of the IgE protein. Here we have analyzed the structural basis of the intrinsic flexibility of this domain. We have compared the sequence of the Cε3 domain to the sequences of other members of the C1 subset of the immunoglobulin superfamily and observed that Cε3 has an unusually high electrostatic charge and an unusually low content of hydrophobic residues. Mutations restoring Cε3 to a more canonical sequence were introduced in an attempt to derive a more structured domain, and several mutants display decreased levels of disorder. Engineered domains of Cε3 with a range of structural rigidities could serve as important tools for the elucidation of the role of flexibility of the Cε3 domain in IgE's biological functions.     

Two distinct variants of erythrocyte spectrin βIV domain

Summary Thirty-nine Turkish phenylketonuria (PKU) families were investigated for their DNA haplotypes at the phenylalanine hydroxylase (PAH) locus. There was a threefold higher incidence of consanguinity in the population studied compared with the general Turkish population. The PAH DNA haplotype 6 was found to be almost exclusively associated not only with the mutant PAH genes but also with the classic phenotype in 39% of the Turkish patients. This haplotype was of no importance in northern European populations. The two DNA haplotypes (1 and 4) that were almost equally frequent among the normal and the mutant PAH genes in northern European populations show virtually the same distribution in Turkish individuals. In all populations studied, these haplotypes are associated with different phenotypes.     

Backbone resonance assignments of the F-actin binding domain of mouse αN-catenin

α-Catenin is a filamentous actin (F-actin) binding protein that links the classical cadherin–catenin complex to the actin cytoskeleton at adherens junctions (AJs). Its C-terminal F-actin binding domain is required for regulating the dynamic interaction between AJs and the actin cytoskeleton during tissue development. Thus, obtaining the molecular details of this interaction is a crucial step towards understanding how α-catenin plays critical roles in biological processes, such as morphogenesis, cell polarity, wound healing and tissue maintenance. Here we report the backbone atom (¹H^N, ¹⁵N, ¹³C^α, ¹³C^β and ¹³C′) resonance assignments of the C-terminal F-actin binding domain of αN-catenin.     

Is the isolated ligand binding domain a good model of the domain in the native receptor?

Numerous studies have used the atomic level structure of the isolated ligand binding domain of the glutamate receptor to elucidate the agonist-induced activation and desensitization processes in this group of proteins. However, no study has demonstrated the structural equivalence of the isolated ligand binding fragments and the protein in the native receptor. In this report, using visible absorption spectroscopy we show that the electronic environment of the antagonist 6-cyano-7-nitro-2,3-dihydroxyquinoxaline is identical for the isolated protein and the native glutamate receptors expressed in cells. Our results hence establish that the local structure of the ligand binding site is the same in the two proteins and validate the detailed structure-function relationships that have been developed based on a comparison of the structure of the isolated ligand binding domain and electrophysiological consequences in the native receptor.     

The PASTA domain: a β-lactam-binding domain

The PASTA domain (for penicillin-binding protein and serine/threonine kinase associated domain) is found in the high molecular weight penicillin-binding proteins and eukaryotic-like serine/threonine kinases of a range of pathogens. We describe this previously uncharacterized domain and infer that it binds β-lactam antibiotics and their peptidoglycan analogues. We postulate that PknB-like kinases are key regulators of cell-wall biosynthesis. The essential function of these enzymes suggests an additional pathway for the action of β-lactam antibiotics.     

Interaction of the α2A domain of integrin with small collagen fragments

We here present a detailed study of the ligand-receptor interactions between single and triple-helical strands of collagen and the α2A domain of integrin (α2A), providing valuable new insights into the mechanisms and dynamics of collagen-integrin binding at a sub-molecular level. The occurrence of single and triple-helical strands of the collagen fragments was scrutinized with atom force microscopy (AFM) techniques. Strong interactions of the triple-stranded fragments comparable to those of collagen can only be detected for the 42mer triple-helical collagen-like peptide under study (which contains 42 amino acid residues per strand) by solid phase assays as well as by surface plasmon resonance (SPR) measurements. However, changes in NMR signals during titration and characteristic saturation transfer difference (STD) NMR signals are also detectable when α2A is added to a solution of the 21mer single-stranded collagen fragment. Molecular dynamics (MD) simulations employing different sets of force field parameters were applied to study the interaction between triple-helical or single-stranded collagen fragments with α2A. It is remarkable that even single-stranded collagen fragments can form various complexes with α2A showing significant differences in the complex stability with identical ligands. The results of MD simulations are in agreement with the signal alterations in our NMR experiments, which are indicative of the formation of weak complexes between single-stranded collagen and α2A in solution. These results provide useful information concerning possible interactions of α2A with small collagen fragments that are of relevance to the design of novel therapeutic A-domain inhibitors.     

NMR structure of the transmembrane domain of the n-acetylcholine receptor β2 subunit

Nicotinic acetylcholine receptors (nAChRs) are involved in fast synaptic transmission in the central and peripheral nervous system. Among the many different types of subunits in nAChRs, the β2 subunit often combines with the α4 subunit to form α4β2 pentameric channels, the most abundant subtype of nAChRs in the brain. Besides computational predictions, there is limited experimental data available on the structure of the β2 subunit. Using high-resolution NMR spectroscopy, we solved the structure of the entire transmembrane domain (TM1234) of the β2 subunit. We found that TM1234 formed a four-helix bundle in the absence of the extracellular and intracellular domains. The structure exhibited many similarities to those previously determined for the Torpedo nAChR and the bacterial ion channel GLIC. We also assessed the influence of the fourth transmembrane helix (TM4) on the rest of the domain. Although secondary structures and tertiary arrangements were similar, the addition of TM4 caused dramatic changes in TM3 dynamics and subtle changes in TM1 and TM2. Taken together, this study suggests that the structures of the transmembrane domains of these proteins are largely shaped by determinants inherent in their sequence, but their dynamics may be sensitive to modulation by tertiary and quaternary contacts.     

Functional role of β domain in the Thermoanaerobacter tengcongensis glucoamylase

Thermoanaerobacter tengcongensis MB4 glucoamylase (TteGA) contains a catalytic domain (CD), which is structurally similar to eukaryotic GA, and a β domain (BD) with ambiguous function. Firstly, BD is found to be essential to TteGA activity because CD alone could not hydrolyze soluble starch. However, starch hydrolysis activity, similar to that of intact TteGA, was restored to CD in the presence of BD. Secondly, BD is found to be an important helper in the correct folding of CD because CD was mainly expressed in the inclusion bodies on its own in Escherichia coli. By contrast, intact TteGA, BD, and CD combined with BD could be expressed as soluble proteins. Additionally, BD is essential to the thermostability of TteGA because CD displayed lower thermostability compared with the intact TteGA and exhibited enhanced thermostability in the presence of BD in vitro. Truncation of TteGA or mutagenesis of the residues that participate in the interdomain interaction at its BD also led to the reduced thermostability of TteGA.     

Controlling conformational flexibility of an O₂-binding H-NOX domain

Heme Nitric oxide/OXygen binding (H-NOX) domains have provided a novel scaffold to probe ligand affinity in hemoproteins. Mutation of isoleucine 5, a conserved residue located in the heme-binding pocket of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX), was carried out to examine changes in oxygen (O(2))-binding properties. A series of I5 mutants (I5F, I5F/I75F, I5F/L144F, I5F/I75F/L144F) were investigated to probe the role of steric bulk within the heme pocket. The mutations significantly increased O(2) association rates (1.5-2.5-fold) and dissociation rates (8-190-fold) as compared to wild-type Tt H-NOX. Structural changes that accompanied the I5F mutation were characterized using X-ray crystallography and resonance Raman spectroscopy. A 1.67 ? crystal structure of the I5F mutant indicated that introducing a phenylalanine at position 5 resulted in a significant shift of the N-terminal domain of the protein, causing an opening of the heme pocket. This movement also resulted in an increased amount of flexibility at the N-terminus and the loop covering the N-terminal helix as indicated by the two conformations of the first six N-terminal amino acids, high B-factors in this region of the protein, and partially discontinuous electron density. In addition, introduction of a phenylalanine at position 5 resulted in increased flexibility of the heme within the pocket and weakened hydrogen bonding to the bound O(2) as measured by resonance Raman spectroscopy. This study provides insight into the critical role of I5 in controlling conformational flexibility and ligand affinity in H-NOX proteins.     

Halophilic characterization of starch-binding domain from Kocuria varians α-amylase

The tandem starch-binding domains (KvSBD) located at carboxy-terminal region of halophilic α-amylase from moderate halophile, Kocuria varians, were expressed in E. coli with amino-terminal hexa-His-tag and purified to homogeneity. The recombinant KvSBD showed binding activity to raw starch granules at low to high salt concentrations. The binding activity of KvSBD to starch was fully reversible after heat-treatment at 85 °C. Circular dichroism and thermal scanning experiments indicated that KvSBD showed fully reversible refolding upon cooling after complete melting at 70 °C in the presence of 0.2-2.0 M NaCl. The refolding rate was enhanced with higher salt concentration.     

Establishment of crown–root domain borders in mouse incisor

Teeth are composed of two domains, the enamel-covered crown and the enamel-free root. The understanding of the initiation and regulation of crown and root domain formation is important for the development of bioengineered teeth. In most teeth the crown develops before the root, and erupts to the oral cavity whereas the root anchors the tooth to the jawbone. However, in the continuously growing mouse incisor the crown and root domains form simultaneously, the crown domain forming the labial and the root domain the lingual part of the tooth. While the crown–root border on the incisor distal side supports the distal enamel extent, reflecting an evolutionary diet adaptation, on the incisor mesial side the root-like surface is necessary for the attachment of the interdental ligament between the two incisors. Therefore, the mouse incisor exhibits a functional distal–mesial asymmetry. Here, we used the mouse incisor as a model to understand the mechanisms involved in the crown–root border formation. We analyzed the cellular origins and gene expression patterns leading to the development of the mesial and distal crown–root borders. We discovered that Barx2, En1, Wnt11, and Runx3 were exclusively expressed on the mesial crown–root border. In addition, the distal border of the crown–root domain might be established by cells from a different origin and by an early Follistatin expression, factor known to be involved in the root domain formation. The use of different mechanisms to establish domain borders gives indications of the incisor functional asymmetry.     

Does domain swapping improve the stability of RNase A?

Self-assembling complexes have potential as novel supramolecular biomaterials but domain swapped complexes have yet to investigated in this capacity. Bovine ribonuclease A (RNase A) is a useful model protein as it is able to form a range of three dimensional domain swapped structures, including dimers, trimers and tetramers that have similar catalytic ability. However, little work has been carried out investigating the physical characteristics of these complexes. In an effort to characterise the strength of these oligomeric interactions, analytical ultracentrifugation was carried out to measure the dissociation of higher order complexes, using fluorescent tags to test for dissociation at very low concentrations. Results of this work suggest that the oligomers form a very tight complex, with no evidence of dissociation down to 250 pM. RNase A oligomers also had similar thermal stability to that of monomeric enzyme, suggesting that the main limiting factor in RNase A stability is the tertiary, rather than quaternary structure. Following thermal unfolding of RNase A, the protein refolded upon cooling, but returned to the monomeric state. This latter result may limit the potential of domain swapping as a means of material assembly.     

Membrane domain organization of myelinated axons requires βII spectrin

The precise and remarkable subdivision of myelinated axons into molecularly and functionally distinct membrane domains depends on axoglial junctions that function as barriers. However, the molecular basis of these barriers remains poorly understood. Here, we report that genetic ablation and loss of axonal βII spectrin eradicated the paranodal barrier that normally separates juxtaparanodal K⁺ channel protein complexes located beneath the myelin sheath from Na⁺ channels located at nodes of Ranvier. Surprisingly, the K⁺ channels and their associated proteins redistributed into paranodes where they colocalized with intact Caspr-labeled axoglial junctions. Furthermore, electron microscopic analysis of the junctions showed intact paranodal septate-like junctions. Thus, the paranodal spectrin-based submembranous cytoskeleton comprises the paranodal barriers required for myelinated axon domain organization.