Modulation of Functional Phosphorylation Sites by Basic Residues in the Unique Domain of c-Src.

In contrast to the well-studied canonical regulatory mechanisms, the way by which the recently discovered Src N-terminal regulatory element (SNRE) modulates Src activity is not yet well understood. Phosphorylation of serine and threonine residues modulates the charge distribution along the disordered region of the SNRE and may affect a fuzzy complex with the SH3 domain that is believed to act as an information transduction element. The pre-existing positively charged sites can interact with the newly introduced phosphate groups by modulating their acidity, introducing local conformational restrictions, or by coupling various phosphosites into a functional unit. In this paper, we use pH-dependent NMR measurements combined with single point mutations to identify the interactions of basic residues with physiologically important phosphorylated residues and to characterize the effect of these interactions in neighbor residues, thus providing insight into the electrostatic network in the isolated disordered regions and in the entire SNRE. From a methodological point of view, the linear relationships observed between the mutation-induced pKa changes of the phosphate groups of phosphoserine and phosphothreonine and the pH-induced chemical shifts of the NH groups of these residues provide a very convenient alternative to identify interacting phosphate groups without the need to introduce point mutations on specific basic residues.

Aß42 assembles into specific ß-barrel pore-forming oligomers in membrane-mimicking environments.

The formation of amyloid-ß peptide (Aß) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in Alzheimer's disease (AD). Therefore, it is critical to characterize the oligomers that form within a membrane environment. To contribute to this characterization, we have applied strategies widely used to examine the structure of membrane proteins to study the two major Aß variants, Aß40 and Aß42. Accordingly, various types of detergent micelles were extensively screened to identify one that preserved the properties of Aß in lipid environments-namely the formation of oligomers that function as pores. Remarkably, under the optimized detergent micelle conditions, Aß40 and Aß42 showed different behavior. Aß40 aggregated into amyloid fibrils, whereas Aß42 assembled into oligomers that inserted into lipid bilayers as well-defined pores and adopted a specific structure with characteristics of a ß-barrel arrangement that we named ß-barrel pore-forming Aß42 oligomers (ßPFOsAß42). Because Aß42, relative to Aß40, has a more prominent role in AD, the higher propensity of Aß42 to form ßPFOs constitutes an indication of their relevance in AD. Moreover, because ßPFOsAß42 adopt a specific structure, this property offers an unprecedented opportunity for testing a hypothesis regarding the involvement of ßPFOs and, more generally, membrane-associated Aß oligomers in AD.

The Two Isoforms of Lyn Display Different Intramolecular Fuzzy Complexes with the SH3 Domain.

The function of the intrinsically disordered Unique domain of the Src family of tyrosine kinases (SFK), where the largest differences between family members are concentrated, remains poorly understood. Recent studies in c-Src have demonstrated that the Unique region forms transient interactions, described as an intramolecular fuzzy complex, with the SH3 domain and suggested that similar complexes could be formed by other SFKs. Src and Lyn are members of a distinct subfamily of SFKs. Lyn is a key player in the immunologic response and exists in two isoforms originating from alternative splicing in the Unique domain. We have used NMR to compare the intramolecular interactions in the two isoforms and found that the alternatively spliced segment interacts specifically with the so-called RT-loop in the SH3 domain and that this interaction is abolished when a polyproline ligand binds to the SH3 domain. These results support the generality of the fuzzy complex formation in distinct subfamilies of SFKs and its physiological role, as the naturally occurring alternative splicing modulates the interactions in this complex.

Active-Site-Directed Inhibitors of Prolyl Oligopeptidase Abolish Its Conformational Dynamics.

Deciphering conformational dynamics is crucial for understanding the biological functions of proteins and for designing compounds targeting them. In particular, providing an accurate description of microsecond-millisecond motions opens the opportunity for regulating protein-protein interactions (PPIs) by modulating the dynamics of one interacting partner. Here we analyzed the conformational dynamics of prolyl oligopeptidase (POP) and the effects of active-site-directed inhibitors on the dynamics. We used an integrated structural biology approach based on NMR spectroscopy and SAXS experiments complemented by MD simulations. We found that POP is in a slow equilibrium in solution between open and closed conformations, and that inhibitors effectively abolished this equilibrium by stabilizing the enzyme in the closed conformation.

An optimized method for (15)N R(1) relaxation rate measurements in non-deuterated proteins.

(15)N longitudinal relaxation rates are extensively used for the characterization of protein dynamics; however, their accurate measurement is hindered by systematic errors. (15)N CSA/(1)H-(15)N dipolar cross-correlated relaxation (CC) and amide proton exchange saturation transfer from water protons are the two main sources of systematic errors in the determination of (15)N R1 rates through (1)H-(15)N HSQC-based experiments. CC is usually suppressed through a train of 180° proton pulses applied during the variable (15)N relaxation period (T), which can perturb water magnetization. Thus CC cancellation is required in such a way as to minimize water saturation effects. Here we examined the level of water saturation during the T period caused by various types of inversion proton pulses to suppress CC: (I) amide-selective IBURP-2; (II) cosine-modulated IBURP-2; (III) Watergate-like blocks; and (IV) non-selective hard. We additionally demonstrate the effect of uncontrolled saturation of aliphatic protons on (15)N R1 rates. In this paper we present an optimized pulse sequence that takes into account the crucial effect of controlling also the saturation of the aliphatic protons during (15)N R1 measurements in non-deuterated proteins. We show that using cosine-modulated IBURP-2 pulses spaced 40 ms to cancel CC in this optimized pulse program is the method of choice to minimize systematic errors coming from water and aliphatic protons saturation effects.

A non-damaging method to analyze the configuration and dynamics of nitrotyrosines in proteins.

Often, deregulation of protein activity and turnover by tyrosine nitration drives cells toward pathogenesis. Hence, understanding how the nitration of a protein affects both its function and stability is of outstanding interest. Nowadays, most of the in vitro analyses of nitrated proteins rely on chemical treatment of native proteins with an excess of a chemical reagent. One such reagent, peroxynitrite, stands out for its biological relevance. However, given the excess of the nitrating reagent, the resulting in vitro modification could differ from the physiological nitration. Here, we determine unequivocally the configuration of distinct nitrated-tyrosine rings in single-tyrosine mutants of cytochrome c. We aimed to confirm the nitration position by a non-destructive method. Thus, we have resorted to (1)H-(15)N heteronuclear single quantum coherence(HSQC) spectra to identify the (3)J(NH) correlation between a (15)N-tagged nitro group and the adjacent aromatic proton. Once the chemical shift of this proton was determined, we compared the (1)H-(13)C HSQC spectra of untreated and nitrated samples. All tyrosines were nitrated at ε positions, in agreement to previous analysis by indirect techniques. Notably, the various nitrotyrosine residues show a different dynamic behaviour that is consistent with molecular dynamics computations.

Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation.

Recent experimental evidence points to intermediates populated during the process of amyloid fibril formation as the toxic moieties primarily responsible for the development of increasingly common disorders such as Alzheimer's disease and type II diabetes. We describe here the application of a pulse-labeling hydrogen-deuterium (HD) exchange strategy monitored by mass spectrometry (MS) and NMR spectroscopy (NMR) to characterize the aggregation process of an SH3 domain under 2 different conditions, both of which ultimately lead to well-defined amyloid fibrils. Under one condition, the intermediates appear to be largely amorphous in nature, whereas under the other condition protofibrillar species are clearly evident. Under the conditions favoring amorphous-like intermediates, only species having no protection against HD exchange can be detected in addition to the mature fibrils that show a high degree of protection. By contrast, under the conditions favoring protofibrillar-like intermediates, MS reveals that multiple species are present with different degrees of HD exchange protection, indicating that aggregation occurs initially through relatively disordered species that subsequently evolve to form ordered aggregates that eventually lead to amyloid fibrils. Further analysis using NMR provides residue-specific information on the structural reorganizations that take place during aggregation, as well as on the time scales by which they occur.

15N relaxation NMR studies of prolyl oligopeptidase, an 80 kDa enzyme, reveal a pre-existing equilibrium between different conformational states.

Open and closed: The characterization of protein mobility is crucial for the understanding of biological functions. We have applied NMR spectroscopy to study the conformational dynamics of the 80 kDa enzyme prolyl oligopeptidase (POP). Our results revealed that POP is highly dynamic and that inhibition of catalytic activity shifts this conformational equilibrium towards a less dynamic state.

Molecular recycling within amyloid fibrils.

Amyloid fibrils are thread-like protein aggregates with a core region formed from repetitive arrays of beta-sheets oriented parallel to the fibril axis. Such structures were first recognized in clinical disorders, but more recently have also been linked to a variety of non-pathogenic phenomena ranging from the transfer of genetic information to synaptic changes associated with memory. The observation that many proteins can convert into similar structures in vitro has suggested that this ability is a generic feature of polypeptide chains. Here we have probed the nature of the amyloid structure by monitoring hydrogen/deuterium exchange in fibrils formed from an SH3 domain using a combination of nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. The results reveal that under the conditions used in this study, exchange is dominated by a mechanism of dissociation and re-association that results in the recycling of molecules within the fibril population. This insight into the dynamic nature of amyloid fibrils, and the ability to determine the parameters that define this behaviour, have important implications for the design of therapeutic strategies directed against amyloid disease.

A Myristoyl-Binding Site in the SH3 Domain Modulates c-Src Membrane Anchoring.

The c-Src oncogene is anchored to the cytoplasmic membrane through its N-terminal myristoylated SH4 domain. This domain is part of an intramolecular fuzzy complex with the SH3 and Unique domains. Here we show that the N-terminal myristoyl group binds to the SH3 domain in the proximity of the RT loop, when Src is not anchored to a lipid membrane. Residues in the so-called Unique Lipid Binding Region modulate this interaction. In the presence of lipids, the myristoyl group is released from the SH3 domain and inserts into the lipid membrane. The fuzzy complex with the SH4 and Unique domains is retained in the membrane-bound form, placing the SH3 domain close to the membrane surface and restricting its orientation. The apparent affinity of myristoylated proteins containing the SH4, Unique, and SH3 domains is modulated by these intramolecular interactions, suggesting a mechanism linking c-Src activation and membrane anchoring.

Side chain to main chain hydrogen bonds stabilize a polyglutamine helix in a transcription factor.

Polyglutamine (polyQ) tracts are regions of low sequence complexity frequently found in transcription factors. Tract length often correlates with transcriptional activity and expansion beyond specific thresholds in certain human proteins is the cause of polyQ disorders. To study the structural basis of the association between tract length, transcriptional activity and disease, we addressed how the conformation of the polyQ tract of the androgen receptor, associated with spinobulbar muscular atrophy (SBMA), depends on its length. Here we report that this sequence folds into a helical structure stabilized by unconventional hydrogen bonds between glutamine side chains and main chain carbonyl groups, and that its helicity directly correlates with tract length. These unusual hydrogen bonds are bifurcate with the conventional hydrogen bonds stabilizing α-helices. Our findings suggest a plausible rationale for the association between polyQ tract length and androgen receptor transcriptional activity and have implications for establishing the mechanistic basis of SBMA.

Preparation of a Well-Defined and Stable ß-Barrel Pore-Forming Aß42 Oligomer.

The formation of amyloid-ß peptide (Aß) oligomers at the cellular membrane is considered a crucial process that underlies neurotoxicity in Alzheimer's disease (AD). To obtain structural information on this type of oligomers, we were inspired by membrane protein approaches used to stabilize, characterize, and analyze the function of such proteins. Using these approaches, we developed conditions under which Aß42, the Aß variant most strongly linked to the aetiology of AD, assembles into an oligomer that inserts into lipid bilayers as a well-defined pore and adopts a specific structure with characteristics of a ß-barrel arrangement. We named this oligomer ß-barrel Pore-Forming Aß42 Oligomer (ßPFOAß42). Here, we describe detailed protocols for its preparation and characterization. We expect ßPFOAß42 to be useful in establishing the involvement of membrane-associated Aß oligomers in AD.

Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism.

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase, SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation.

NMR studies of protein-ligand interactions.

Nuclear magnetic resonance (NMR) has evolved into a powerful tool for characterizing protein-ligand interactions in solution under near physiological conditions. It is now frequently harnessed to assess the affinity and specificity of interactions; to identify binding epitopes on proteins and ligands; and to characterize the structural rearrangements induced by binding.The first section of this chapter provides a general overview of the NMR study of protein-ligand interactions. The section is divided according to two main categories of experiments: those based on observing protein signals and those based on observing ligand signals. The next section explains two case studies performed in the authors' laboratory. The first of these deals with the interaction between vascular endothelial growth factor and a peptidic ligand, and includes a detailed protocol of chemical shift perturbation experiments. The second one reports on the interaction between prolyl oligopeptidase and a small molecule as monitored by ligand saturation transfer difference (STD), and illustrates how NMR can be used to confirm binding and to identify the binding epitope of a ligand.

Structural characterization of the natively unfolded N-terminal domain of human c-Src kinase: insights into the role of phosphorylation of the unique domain.

The N-terminal regions of the members of Src family of non-receptor protein tyrosine kinases are intrinsically unfolded and contain the maximum sequence divergence among them. In this study, we have addressed the structural characterization by nuclear magnetic resonance of this region of 84 residues that encompasses the SH4 and the unique domains (USrc) of the human c-Src. With this aim, the backbone assignment was performed using (13)C-detected experiments that overcome the spectral resolution problems and the large number of prolines that are typical for intrinsically unfolded proteins. The analysis of the residual dipolar couplings measured for the USrc indicates the presence of a low populated helical structure in the 60-75 region. No long-range contacts between remote fragments of the chain were detected with paramagnetic relaxation enhancement experiments. The structural characterization was extended to two different phosphorylation states of USrc that encompassed three different phosphorylated sites, Ser17, Thr37, and Ser75. The structural and conformational changes upon phosphorylation were monitored through chemical shift perturbations and residual dipolar couplings, indicating that modifications occur at local level and no global rearrangements were apparent. These results suggest a scenario where phosphorylation induces a global electrostatic perturbation that could be involved in the membrane unbinding of c-Src and that could be related with the localization of the enzyme. These observations suggest the unique domain of Src kinases as a source of selectivity and reinforce the relevant role of intrinsically disordered proteins in biological processes.

A new side opening on prolyl oligopeptidase revealed by electron microscopy.

Prolyl oligopeptidase (POP) has gained importance as a target for the treatment of neuropsychiatric diseases and cognitive disturbances. Therefore, a variety of strategies are currently used to identify POP inhibitors. Here we performed electron microscopy (EM) studies of human POP. Our data reveal for the first time the presence of a new side opening in POP that was not observed in any of the crystallographic structures described to date. Finally, molecular dynamics, the relevant normal modes that contribute to the fluctuation of the catalytic triad residues and the algorithm CAVERN also support the existence of a new large side opening on POP.

Conformational analysis of a potent SSTR3-selective somatostatin analogue by NMR in water solution.

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2). The structural determinant factors that could explain selectivity of peptide 1 for SSTR3 receptors are discussed.

Dynamic properties of biologically active synthetic heparin-like hexasaccharides.

A complete study of the dynamics of two synthetic heparin-like hexasaccharides, D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-1-->iPr (1) and -->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHAc-6-SO4-alpha-(1-->4)-L-IdoA-alpha-(1-->4)-D-GlcNHSO3-alpha-(1-->4)-L-IdoA-2-SO4-alpha-1-->iPr (2), has been performed using 13C-nuclear magnetic resonance (NMR) relaxation parameters, T1, T2, and heteronuclear nuclear Overhauser effect (NOEs). Compound 1 is constituted from sequences corresponding to the major polysaccharide heparin region, while compound 2 contains a sequence never found in natural heparin. They differ from each other only in sulphation patterns, and are capable of stimulating fibroblast growth factors (FGFs)-1 induced mitogenesis. Both oligosaccharides exhibit a remarkable anisotropic overall motion in solution as revealed by their anisotropic ratios (tau /tau||), 4.0 and 3.0 respectively. This is a characteristic behaviour of natural glycosaminoglycans (GAG) which has also been observed for the antithrombin (AT) binding pentasaccharide D-GlcNHSO3-6-SO4-alpha-(1-->4)-D-GlcA-beta-(1-->4)-D-GlcNHSO3-(3,6-SO4)-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-1-->Me (3) (Hricovíni, M., Guerrini, M., Torri, G., Piani, S., and Ungarelli, F. (1995) Conformational analysis of heparin epoxide in aqueous solution. An NMR relaxation study. Carbohydr. Res., 277, 11-23). The motional properties observed for 1 and 2 provide additional support to the suitability of these compounds as heparin models in agreement with previous structural (de Paz, J.L., Angulo, J., Lassaletta, J.M., Nieto, P.M., Redondo-Horcajo, M., Lozano, R.M., Jiménez-Gallego, G., and Martín-Lomas, M. (2001) The activation of fibroblast growth factors by heparin: synthesis, structure and biological activity of heparin-like oligosaccharides. Chembiochem, 2, 673-685; Ojeda, R., Angulo, J., Nieto, P.M., and Martin-Lomas. M. (2002) The activation of fibroblast growth factors by heparin: synthesis and structural study of rationally modified heparin-like oligosaccharides. Can. J. Chem,. 80, 917-936; Lucas, R., Angulo, J., Nieto, P.M., and Martin-Lomas, M. (2003) Synthesis and structural studies of two new heparin-like hexasaccharides. Org. Biomol. Chem., 1, 2253-2266) and biological data (Angulo, J., Ojeda, R., de Paz, J.L., Lucas, R., Nieto, P.M., Lozano, R.M., Redondo-Horcajo, M., Giménez-Gallego, G., and Martín-Lomas, M. (2004) The activation of fibroblast growth factors (FGFs) by glycosaminoglycans: influence of the sulphation pattern on the biological activity of FGF-1. Chembiochem, 5, 55-61). Fast internal motions observed for the less sulphated compound 2, as compared with 1, may be related to their different behavior in stimulating FGF1-induced mitogenic activity.