Structural basis for ubiquitin recognition by SH3 domains

The SH3 domain is a protein-protein interaction module commonly found in intracellular signaling and adaptor proteins. The SH3 domains of multiple endocytic proteins have been recently implicated in binding ubiquitin, which serves as a signal for diverse cellular processes including gene regulation, endosomal sorting, and protein destruction. Here we describe the solution NMR structure of ubiquitin in complex with an SH3 domain belonging to the yeast endocytic protein Sla1. The ubiquitin binding surface of the Sla1 SH3 domain overlaps substantially with the canonical binding surface for proline-rich ligands. Like many other ubiquitin-binding motifs, the SH3 domain engages the Ile44 hydrophobic patch of ubiquitin. A phenylalanine residue located at the heart of the ubiquitin-binding surface of the SH3 domain serves as a key specificity determinant. The structure of the SH3-ubiquitin complex explains how a subset of SH3 domains has acquired this non-traditional function.     

Thermodynamics of binding to SH3 domains: the energetic impact of polyproline II (PII) helix formation

Although numerous biophysical studies have focused on elucidating the structural and thermodynamic determinants that govern the free energy of binding between various SH3 domains and their putative recognition sequences, a quantitative accounting of the energetics of this interaction has proven enigmatic. Specifically, the binding results in a large and negative change on the standard enthalpy and entropy functions, a result which is inconsistent with the positive values for these quantities that is expected from the hydrophobic nature of the binding pocket. Here, the binding of the C-terminal SH3 domain of Sem-5 to its putative recognition peptide on the Sos (Son of Sevenless) protein is investigated using isothermal titration calorimetry under a variety of temperature and pH conditions. In addition, the energy associated with folding the Sos peptide into the binding competent polyproline II conformation is quantitatively evaluated. These results provide a rationale for the observed discrepancy between the experimental and predicted behavior and indicate that the determinants of binding in this system cannot be ascertained from a static structural representation of the binding process.     

Solution structure of N-terminal SH3 domain of Vav and the recognition site for Grb2 C-terminal SH3 domain

The three-dimensional structure of the N-terminal SH3 domain (residues 583–660) of murine Vav, which contains a tetra-proline sequence (Pro 607-Pro 610), was determined by NMR. The solution structure of the SH3 domain shows a typical SH3 fold, but it exists in two conformations due to cis-trans isomerization at the Gly614-Pro615 bond. The NMR structure of the P615G mutant, where Pro615 is replaced by glycine, reveals that the tetra-proline region is inserted into the RT-loop and binds to its own SH3 structure. The C-terminal SH3 domain of Grb2 specifically binds to the trans form of the N-terminal SH3 domain of Vav. The surface of Vav N-terminal SH3 which binds to Grb2 C-terminal SH3 was elucidated by chemical shift mapping experiments using NMR. The surface does not involve the tetra-proline region but involves the region comprising the n-src loop, the N-terminal and the C-terminal regions. This surface is located opposite to the tetra-proline containing region, consistent with that of our previous mutagenesis studies.     

Sequence-specific minor groove binding by bis-benzimidazoles: water molecules in ligand recognition

The binding of two symmetric bis-benzimidazole compounds, 2,2-bis-[4′-(3″-dimethylamino-1″-propyloxy)phenyl]-5,5-bi-1H-benzimidazole and its piperidinpropylphenyl analog, to the minor groove of DNA, have been studied by DNA footprinting, surface plasmon resonance (SPR) methods and molecular dynamics simulations in explicit solvent. The footprinting and SPR methods find that the former compound has enhanced affinity and selectivity for AT sequences in DNA. The molecular modeling studies have suggested that, due to the presence of the oxygen atom in each side chain of the former compound, a water molecule is immobilized and effectively bridges between side chain and DNA base edges via hydrogen bonding interactions. This additional contribution to ligand–DNA interactions would be expected to result in enhanced DNA affinity, as is observed.     

Enthalpy changes for hemoglobin-ligand interactions: revised calorimetric data

Recently we reported values for the succesive enthalpy changes for the four steps of oxygen binding by diphosphoglycerate-free adult human hemoglobin [Biochem. Biophys. Res. Comm., $\text{56}$, 555 (1974)]. Systematic errors in the data render these results invalid. New data shows that cooperativity prevents resolution of successive heats of reaction and only average heats are currently accessible. At pH 7.4 and 6^o we obtain: $\text{Δ}\text{H[}\text{1}\text{4}\text{Hb+O}{}_2\text{→}\text{1}\text{4}\text{Hb (O}{}_2\text{)}{}_4\text{]=?13.2±0.4}$, $\text{Δ}\text{H[}\text{1}\text{4}\text{Hb(O}{}_2\text{)}{}_4\text{+CO→}\text{1}\text{4}\text{Hb(CO)}{}_4\text{+O}{}_2\text{]=?4.0±0.1}$, $\text{Δ}\text{H[}\text{1}\text{4}\text{Hb+CO→}\text{1}\text{4}\text{Hb(CO)}{}_4\text{] =?17.7±0.4 kcal/mole}$ ligand. These data form a consistent thermodynamic cycle.     

Physical water compartments: a revised concept of perinatal body water physiology

This review presents experimental data on the perinatal significance of the recently developed concept of physical water compartments. This concept implies that in addition to the compartmentalization of body water into the intra- and extracellular spaces, motionally distinct water fractions - designated as physical water compartments - are also of importance in the neonatal body fluid redistribution. H(1)-NMR spectroscopy provides a quantitative estimate of tissue water fractions with different mobility as multicomponent analysis of the T(2) relaxation decay curves allows us to determine the fast and slow relaxing components of the curves corresponding to the bound and free fractions of tissue water. Using this method, free and bound water fractions were measured in fetal and neonatal rabbit tissues (skin, skeletal muscle, liver, brain, lung) at different stages of maturity and under conditions of various fluid intake. It has been demonstrated that water mobility in individual fetal/neonatal tissues varies greatly and there is a general tendency of increasing free water at the expense of bound water fraction with progressing maturation. This tendency appears to be accelerated in the immediate postnatal period when the tissue water content is markedly reduced. The importance of hyaluronan in this process has also been addressed as the hyaluronan content is markedly elevated in the fetal/neonatal tissues and due to its polyanionic, hydrophilic nature it has been claimed to play a prominent but not clearly defined role in the control of tissue hydration.     

Motor cognition: a new paradigm to study self-other interactions

Accumulative empirical evidence has been reviewed in support of the notion that the production and perception of action as well as the interpretation of others' actions are functionally connected, and indeed, rely on common distributed neural systems in the premotor and parietal cortices. We suggest that these neural systems sustain shared representations between self and other that are crucial in social interactions. The inferior parietal cortex plays a special role in the sense of agency, which is a fundamental aspect to navigate within this neural network. The role of other brain areas that implement and regulate these shared representations remains to be specified.     

Crosstalk between cell adhesion molecules: vinculin as a paradigm for regulation by conformation

With the identification of ever more protein components associated with cellular adhesion sites, the nature of the mechanisms underlying assembly and maintenance of these important cellular structures was in danger of becoming completely intangible. However, new information on how the interaction between the different proteins can be regulated is beginning to shed more light on this problem. In particular, recent biochemical and electron microscopic data on the overall structure and function of vinculin, one of the key structural proteins involved in cellular adhesion, leads to a novel model for the regulation of cellular adhesion.     

Novartis Medal Lecture. Antibodies: a paradigm for the evolution of molecular recognition

Novel proteins have been elaborated over evolutionary time by an iterative alternation of mutation and selection. In a similar way, the humoral immune system also uses an iterative alternation of mutation and selection to generate novel antibodies that display a high affinity for their cognate antigen -- but this is achieved in a matter of a days. Gene rearrangement is used to produce a primary repertoire of antibodies and, on entering the body, antigen triggers the clonal expansion of those B lymphocytes that express a cognate antibody, albeit one of low affinity. Rapid and specific affinity maturation is then achieved by subjecting the immunoglobulin genes in the rapidly expanding B cells to a period of intense mutation. The intensity of this mutational assault is tolerated because it is targeted specifically to the immunoglobulin genes, causing relatively little damage to other loci. Antigen-mediated selection then allows the preferential expansion of those mutants expressing antibodies displaying improved binding characteristics. Here, studies are described that have been performed to glean insight into the mechanisms of the hypermutation and selection processes. Experiments are also described in which an attempt has been made to recapitulate aspects of physiological antibody generation in vitro, allowing the development of novel approaches to the generation of proteins with high-affinity binding sites.     

Structure of the Homer EVH1 domain-peptide complex reveals a new twist in polyproline recognition

Homer EVH1 (Ena/VASP Homology 1) domains interact with proline-rich motifs in the cytoplasmic regions of group 1 metabotropic glutamate receptors (mGluRs), inositol-1,4,5-trisphosphate receptors (IP3Rs), and Shank proteins. We have determined the crystal structure of the Homer EVH1 domain complexed with a peptide from mGluR (TPPSPF). In contrast to other EVH1 domains, the bound mGluR ligand assumes an unusual conformation in which the side chains of the Ser-Pro tandem are oriented away from the Homer surface, and the Phe forms a unique contact. This unusual binding mode rationalizes conserved features of both Homer and Homer ligands that are not shared by other EVH1 domains. Site-directed mutagenesis confirms the importance of specific Homer residues for ligand binding. These results establish a molecular basis for understanding the biological properties of Homer-ligand complexes.     

Tripeptides with ionizable side chains adopt a perturbed polyproline II structure in water

The present paper reports the conformations of the acidic and basic homotripeptides triglutamate, triaspartate, and trilysine in aqueous solution to better understand their relevance for the structure of disordered proteins and protein segments and for a variety of protein binding processes. The determination of the dihedral angles of the central amino acid residue was achieved by analyzing the amide I band profile of the respective polarized visible Raman, Fourier transform infrared (FT-IR), and vibrational circular dichroism (VCD) spectra by means of recently developed algorithms [Schweitzer-Stenner, R. (2002) Biophys. J. 83, 523-532; Eker et al. (2002) J. Am. Chem. Soc. 124, 523-532]. The results were validated by measuring the UV electronic circular dichroism (ECD) spectra of the peptides. The analyses revealed that a polyproline II-like conformation is predominant at room temperature. For triaspartate and triglutamate the dihedral angles of phi = -70 degrees, psi = 165 degrees and phi = -60 degrees, psi = 160 degrees were obtained, respectively. A similar conformation, i.e., phi = -50 degrees, psi = 170 degrees, was obtained for trilysine, which is at variance with the earlier reported left-handed turn structure. The ECD spectrum of charged tripeptides displayed symmetric negative and positive couplets at 190 and 210 nm, which are interpreted as indicating a somewhat, perturbed polyproline II conformation, in agreement with the obtained dihedral angles. Comparison with literature data shows that the investigated tripeptides are ideal model systems for understanding the local conformation of functionally relevant K3, K2X, E3, and D3 segments in a variety of different proteins.     

Cooperative interactions and a non-native buried Trp in the unfolded state of an SH3 domain

The presence of residual structure in the unfolded state of the N-terminal SH3 domain of Drosophila drk (drkN SH3 domain) has been investigated using far- and near-UV circular dichroism (CD), fluorescence, and NMR spectroscopy. The unfolded (U(exch)) state of the drkN SH3 domain is significantly populated and exists in equilibrium with the folded (F(exch)) state under non-denaturing conditions near physiological pH. Denaturation experiments have been performed on the drkN SH3 domain in order to monitor the change in ellipticity, fluorescence intensity, and chemical shift between the U(exch) state and chemically or thermally denatured states. Differences between the unfolded and chemically or thermally denatured states highlight specific areas of residual structure in the unfolded state that are cooperatively disrupted upon denaturation. Results provide evidence for cooperative interactions in the unfolded state involving residues of the central beta-sheet, particularly the beta4 strand. Denaturation as well as hydrogen-exchange experiments demonstrate a non-native burial of the Trp ring within this "cooperative" core of the unfolded state. These findings support the presence of non-native hydrophobic clusters, organised by Trp rings, within disordered states.     

How tetrapods feed in water: a functional analysis by paradigm

Mechanical theory is used to erect a paradigm predicting the manipulations used by carnivorous aquatic amphibians, reptiles, birds and mammals to catch, subdue, process and swallow their prey. These predictions are confirmed by observational evidence. Most aquatic predatory tetrapods use long, prehensile tooth-armed jaws as pincer jaws to snap shut onto the prey and catch and kill it, although some use the flexibility of long necks in spear fishing and some odontocetes may stun prey with sonar. Most do not have cutting or nipping dentitions as these cannot be used on prey which is freely floating. They use caniniform dentition to hold and kill prey, or in some cases crushing dentition to break open hard-shelled prey. They dismember prey by dynamic loading, snatching bites so quickly that the prey tears. They use shake feeding, shaking the prey apart from side to side above the water. If the prey is too large to lift above the water they use twist feeding, twisting pieces off. Small pieces are easily swallowed but larger pieces are held above the water and swallowed by tilting the head back in gravity feeding, or by jerking the head back and forth in incrtial feeding. Some animals use mobile jaws to pull prey back into the mouth in ratchet feeding. Filter feeding evades these problems by feeding on very small prey. The use of paradigms in functional analysis is discussed with special reference to this work. The paradigm method is shown to be the most suitable one. There has been repeated convergent and parallel evolution of adaptations to feed in water.     

Nonantibody-based recognition: alternative molecules for detection of pathogens

Immunoassays have been well established for many years as the cornerstone of detection technologies. These assays are sensitive, selective and, in general, highly resistant to interference from complex sample matrices when compared with nucleic acid-based tests. However, both antibody- and nucleic acid-based detection systems require a priori knowledge of the target and development of specific reagents; multiplexed assays can become increasingly problematic when attempting to detect a plethora of different targets, the identities of which are unknown. In an effort to circumvent many of the limitations inherent in these conventional assays, other recognition reagents are being explored as alternatives, or indeed as adjuncts, to antibodies for pathogen and toxin detection. This article will review a number of different recognition systems ranging in complexity from small molecules, such as nucleic-acid aptamers, carbohydrates and peptides, to systems as highly complicated as whole cells and organisms. All of these alternative systems have tremendous potential to achieve superior sensitivity, selectivity, and stability, but are also subject to their own limitations, which are also discussed. In short, while in its infancy, this field holds great promise for the development of rapid, fieldable assays that are highly complementary to existing antibody- and nucleic acid-based technologies.     

Elucidation of the binding preferences of peptide recognition modules: SH3 and PDZ domains

Peptide-binding domains play a critical role in regulation of cellular processes by mediating protein interactions involved in signalling. In recent years, the development of large-scale technologies has enabled exhaustive studies on the peptide recognition preferences for a number of peptide-binding domain families. These efforts have provided significant insights into the binding specificities of these modular domains. Many research groups have taken advantage of this unprecedented volume of specificity data and have developed a variety of new algorithms for the prediction of binding specificities of peptide-binding domains and for the prediction of their natural binding targets. This knowledge has also been applied to the design of synthetic peptide-binding domains in order to rewire protein-protein interaction networks. Here, we describe how these experimental technologies have impacted on our understanding of peptide-binding domain specificities and on the elucidation of their natural ligands. We discuss SH3 and PDZ domains as well characterized examples, and we explore the feasibility of expanding high-throughput experiments to other peptide-binding domains.     

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3''-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha.     

Regulatory role of SH3 domain-mediated protein-protein interactions in synaptic vesicle endocytosis.

Src homology (SH) 3 domains are small modules found in a diverse array of proteins. The presence of an SH3 domain confers upon its resident protein the ability to interact with specific proline-rich sequences in protein binding partners. A major focus of research has highlighted a role for SH3 domain-mediated interactions in the regulation of signal transduction events. However, more recent data has suggested an important function for SH3 domains in vesicular trafficking. This review will focus on this newly emerging role with a particular emphasis on the molecular components involved in synaptic vesicle endocytosis and the regulatory role of SH3 domain-mediated protein-protein interactions in this process.     

Distinct interactions between ubiquitin and the SH3 domains involved in immune signaling

The SH3 domain is a versatile protein interaction motif that generally recognizes proline rich sequences (PRS). Recently, it has been shown that some SH3 domains in the endocytotic pathway can bind to ubiquitin. Moreover, Phe73 in the SH3 domain has been proposed to be an important determinant of the interaction, as the SH3 domains having Tyr73, either naturally or by mutation, failed to bind. Since SH3 domains are also important in immune receptor signaling, we investigated the interactions between immunologically relevant SH3 domains and ubiquitin. We observed that some of these SH3 domains can also bind to ubiquitin. Interestingly, we found that Nck2-SH3-3 bound to ubiquitin despite its Tyr at residue 73 (Tyr56 in our actual construct), but that CD2BP1-SH3 failed to bind, even though it has Phe at an equivalent position. Through detailed NMR binding studies on SH3 domains with Phes and Tyrs at the 73 position, we found that the two types of SH3 domains exhibit mechanistic differences in ubiquitin binding. We showed that the relative contribution of each binding sub-region in both SH3 domains and ubiquitin is quite different in the two binding modes. Such results raise the possibility that the mechanistic variety of these immunologically relevant SH3 domains might contribute to their functional diversity.