Protein interacting with C-kinase 1 (PICK1) binding promiscuity relies on unconventional PSD-95/discs-large/ZO-1 homology (PDZ) binding modes for nonclass II PDZ ligands.

PDZ domain proteins control multiple cellular functions by governing assembly of protein complexes. It remains unknown why individual PDZ domains can bind the extreme C terminus of very diverse binding partners and maintain selectivity. By employing NMR spectroscopy, together with molecular modeling, mutational analysis, and fluorescent polarization binding experiments, we identify here three structural mechanisms explaining why the PDZ domain of PICK1 selectively binds >30 receptors, transporters, and kinases. Class II ligands, including the dopamine transporter, adopt a canonical binding mode with promiscuity obtained via differential packing in the binding groove. Class I ligands, such as protein kinase Cα, depend on residues upstream from the canonical binding sequence that are likely to interact with flexible loop residues of the PDZ domain. Finally, we obtain evidence that the unconventional ligand ASIC1a has a dual binding mode involving a canonical insertion and a noncanonical internal insertion with the two C-terminal residues forming interactions outside the groove. Together with an evolutionary analysis, the data show how unconventional binding modes might evolve for a protein recognition domain to expand the repertoire of functionally important interactions.

Exploring the minimally frustrated energy landscape of unfolded ACBP.

The unfolded state of globular proteins is not well described by a simple statistical coil due to residual structural features, such as secondary structure or transiently formed long-range contacts. The principle of minimal frustration predicts that the unfolded ensemble is biased toward productive regions in the conformational space determined by the native structure. Transient long-range contacts, both native-like and non-native-like, have previously been shown to be present in the unfolded state of the four-helix-bundle protein acyl co-enzyme binding protein (ACBP) as seen from both perturbations in nuclear magnetic resonance (NMR) chemical shifts and structural ensembles generated from NMR paramagnetic relaxation data. To study the nature of the contacts in detail, we used paramagnetic NMR relaxation enhancements, in combination with single-point mutations, to obtain distance constraints for the acid-unfolded ensemble of ACBP. We show that, even in the acid-unfolded state, long-range contacts are specific in nature and single-point mutations affect the free-energy landscape of the unfolded protein. Using this approach, we were able to map out concerted, interconnected, and productive long-range contacts. The correlation between the native-state stability and compactness of the denatured state provides further evidence for native-like contact formation in the denatured state. Overall, these results imply that, even in the earliest stages of folding, ACBP dynamics are governed by native-like contacts on a minimally frustrated energy landscape.

Modulation of the intrinsic helix propensity of an intrinsically disordered protein reveals long-range helix-helix interactions.

Intrinsically disordered proteins (IDPs) are widespread and important in biology but defy the classical protein structure-function paradigm by being functional in the absence of a stable, folded conformation. Here we investigate the coupling between transient secondary and tertiary structure in the protein activator for thyroid hormone and retinoid receptors (ACTR) by rationally modulating the helical propensity of a partially formed α-helix via mutations. Eight mutations predicted to affect the population of a transient helix were produced and investigated by NMR spectroscopy. Chemical shift changes distant to the mutation site are observed in regions containing other transient helices indicating that distant helices are stabilized through long-range hydrophobic helix-helix interactions and demonstrating the coupling of transient secondary and tertiary structure. The long-range structure of ACTR is also probed using paramagnetic relaxation enhancements (PRE) and residual dipolar couplings, which reveal an additional long-range contact between the N- and C-terminal segments. Compared to residual dipolar couplings and PRE, modulation of the helical propensity by mutagenesis thus reveals a different set of long-range interactions that may be obscured by stronger interactions that dominate other NMR measurements. This approach thus offers a complementary and generally applicable strategy for probing long-range structure in disordered proteins.

Solution properties of the archaeal CRISPR DNA repeat-binding homeodomain protein Cbp2.

Clustered regularly interspaced short palindromic repeats (CRISPR) form the basis of diverse adaptive immune systems directed primarily against invading genetic elements of archaea and bacteria. Cbp1 of the crenarchaeal thermoacidophilic order Sulfolobales, carrying three imperfect repeats, binds specifically to CRISPR DNA repeats and has been implicated in facilitating production of long transcripts from CRISPR loci. Here, a second related class of CRISPR DNA repeat-binding protein, denoted Cbp2, is characterized that contains two imperfect repeats and is found amongst members of the crenarchaeal thermoneutrophilic order Desulfurococcales. DNA repeat-binding properties of the Hyperthermus butylicus protein Cbp2Hb were characterized and its three-dimensional structure was determined by NMR spectroscopy. The two repeats generate helix-turn-helix structures separated by a basic linker that is implicated in facilitating high affinity DNA binding of Cbp2 by tethering the two domains. Structural studies on mutant proteins provide support for Cys(7) and Cys(28) enhancing high thermal stability of Cbp2Hb through disulphide bridge formation. Consistent with their proposed CRISPR transcriptional regulatory role, Cbp2Hb and, by inference, other Cbp1 and Cbp2 proteins are closely related in structure to homeodomain proteins with linked helix-turn-helix (HTH) domains, in particular the paired domain Pax and Myb family proteins that are involved in eukaryal transcriptional regulation.

Using guanidine-hydrochloride for fast and efficient protein digestion and single-step affinity-purification mass spectrometry.

Protein digestion is an integral part of the "shotgun" proteomics approach and commonly requires overnight incubation prior to mass spectrometry analysis. Quadruplicate "shotgun" proteomic analysis of whole yeast lysate demonstrated that Guanidine-Hydrochloride (Gnd-HCl) protein digestion can be optimally completed within 30 min with endoprotease Lys-C. No chemical artifacts were introduced when samples were incubated in Gnd-HCl at 95 °C, making Gnd-HCl an appropriate digestion buffer for shotgun proteomics. Current methodologies for investigating protein-protein interactions (PPIs) often require several preparation steps, which prolongs any parallel operation and high-throughput interaction analysis. Gnd-HCl allow the efficient elution and subsequent fast digestion of PPIs to provide a convenient high-throughput methodology for affinity-purification mass spectrometry (AP-MS) experiments. To validate the Gnd-HCl approach, label-free PPI analysis of several GFP-tagged yeast deubiquitinating enzymes was performed. The identification of known interaction partners demonstrates the utility of the optimized Gnd-HCl protocol that is also scalable to the 96 well-plate format.

GAGE cancer-germline antigens are recruited to the nuclear envelope by germ cell-less (GCL).

GAGE proteins are highly similar, primate-specific molecules with unique primary structure and undefined cellular roles. They are restricted to cells of the germ line in adult healthy individuals, but are broadly expressed in a wide range of cancers. In a yeast two-hybrid screen we identified the metazoan transcriptional regulator, Germ cell-less (GCL), as an interaction partner of GAGE12I. GCL directly binds LEM-domain proteins (LAP2ß, emerin, MAN1) at the nuclear envelope, and we found that GAGE proteins were recruited to the nuclear envelope inner membrane by GCL. Based on yeast two-hybrid analysis and pull-down experiments of GCL polypeptides, GCL residues 209-320 (which includes the BACK domain) were deduced sufficient for association with GAGE proteins. GAGE mRNAs and GCL mRNA were demonstrated in human testis and most types of cancers, and at the protein level GAGE members and GCL were co-expressed in cancer cell lines. Structural studies of GAGE proteins revealed no distinct secondary or tertiary structure, suggesting they are intrinsically disordered. Interestingly GAGE proteins formed stable complexes with dsDNA in vitro at physiological concentrations, and GAGE12I bound several different dsDNA fragments, suggesting sequence-nonspecific binding. Dual association of GAGE family members with GCL at the nuclear envelope inner membrane in cells, and with dsDNA in vitro, implicate GAGE proteins in chromatin regulation in germ cells and cancer cells.

Nectin-1 binds and signals through the fibroblast growth factor receptor.

Nectins belong to a family of immunoglobulin (Ig)-like cell-adhesion molecules comprising four members, nectin-1 through nectin-4. Nectins are involved in formation of the mechanical adhesive puncta adherentia junctions of synapses. Nectins share the same overall structural topology with an extracellular region containing three Ig modules, a transmembrane region, and a cytoplasmic region. In nectin-1, the first and second Ig module in the extracellular region are necessary for the trans-interaction with nectin-3 and formation of cis-dimers, respectively. The function of the third Ig module of nectin-1 remains unknown. We here report the structure in solution of the third, membrane-proximal Ig module of mouse nectin-1 (nectin-1 Ig3) solved by means of nuclear magnetic resonance (NMR) spectroscopy. It belongs to the C1 set of the Ig superfamily. Nectin-1 Ig3 was produced as a recombinant protein and induced neurite outgrowth in primary cultures of hippocampal and cerebellar granule neurons, an effect abolished by treatment with the fibroblast growth factor receptor (FGFR) inhibitor SU5402, or by transfection with a dominant-negative FGFR1 construct. We showed by surface plasmon resonance (SPR) analysis that nectin-1 Ig3 directly interacted with various isoforms of FGFR. Nectin-1 Ig3 induced phosphorylation of FGFR1c in the same manner as the whole nectin-1 ectodomain, and promoted survival of cerebellar granule neurons induced to undergo apoptosis. Finally, we constructed a peptide, nectide, by employing in silico modeling of various FGFR ligand-binding sites. Nectide mimicked all the effects of nectin-1 Ig3. We suggest that FGFR is a downstream signaling partner of nectin-1.

A highly compliant protein native state with a spontaneous-like mechanical unfolding pathway.

The mechanical properties of proteins and their force-induced structural changes play key roles in many biological processes. Previous studies have shown that natively folded proteins are brittle under tension, unfolding after small mechanical deformations, while partially folded intermediate states, such as molten globules, are compliant and can deform elastically a great amount before crossing the transition state barrier. Moreover, under tension proteins appear to unfold through a different sequence of events than during spontaneous unfolding. Here, we describe the response to force of the four-α-helix acyl-CoA binding protein (ACBP) in the low-force regime using optical tweezers and ratcheted molecular dynamics simulations. The results of our studies reveal an unprecedented mechanical behavior of a natively folded protein. ACBP displays an atypical compliance along two nearly orthogonal pulling axes, with transition states located almost halfway between the unfolded and folded states. Surprisingly, the deformability of ACBP is greater than that observed for the highly pliant molten globule intermediate states. Furthermore, when manipulated from the N- and C-termini, ACBP unfolds by populating a transition state that resembles that observed during chemical denaturation, both for structure and position along the reaction coordinate. Our data provide the first experimental evidence of a spontaneous-like mechanical unfolding pathway of a protein. The mechanical behavior of ACBP is discussed in terms of topology and helix propensity.

Temperature-induced transitions in disordered proteins probed by NMR spectroscopy.

Intrinsically disordered proteins are abundant in nature and perform many important physiological functions. Multidimensional NMR spectroscopy has been crucial for the understanding of the conformational properties of disordered proteins and is increasingly used to probe their conformational ensembles. Compared to folded proteins, disordered proteins are more malleable and more easily perturbed by environmental factors. Accordingly, the experimental conditions and especially the temperature modify the structural and functional properties of disordered proteins. NMR spectroscopy allows analysis of temperature-induced structural changes at residue resolution using secondary chemical shift analysis, paramagnetic relaxation enhancement, and residual dipolar couplings. This chapter discusses practical aspects of NMR studies of temperature-induced structural changes in disordered proteins.

Analyzing temperature-induced transitions in disordered proteins by NMR spectroscopy and secondary chemical shift analyses.

Intrinsically disordered proteins are abundant in nature and perform many important physiological functions. Multidimensional NMR spectroscopy has been crucial for the understanding of the conformational properties of disordered proteins and is increasingly used to probe their conformational ensembles. Compared to folded proteins, disordered proteins are more malleable and more easily perturbed by environmental factors. Accordingly, the experimental conditions and especially the temperature modify the structural and functional properties of disordered proteins. This chapter discusses practical aspects of NMR studies of temperature-induced structural changes in disordered proteins using chemical shifts.

Is a malleable protein necessarily highly dynamic? The hydrophobic core of the nuclear coactivator binding domain is well ordered.

The nuclear coactivator binding domain of CREB binding protein folds into remarkably different structures in complex with different ligands. To understand the mechanism of the structural adaptability in the nuclear coactivator binding domain (NCBD), we have compared the dynamics of the hydrophobic core of NCBD in the ligand-free state and in a well-folded complex with the ligand activator for thyroid hormone and retinoid receptors using multiple NMR methods including methyl chemical shifts, coupling constants, and methyl order parameters. From all NMR measures, the aliphatic side chains in the hydrophobic core are slightly more dynamic in the free protein than in the complex, but have mobility comparable to the hydrophobic cores of average folded proteins. Urea titration monitored by NMR reveals that all parts of the protein, including the side-chain packing in the hydrophobic core, denatures in a single cooperative process. The molten globule characteristics of NCBD are thus restricted to a slowly fluctuating tertiary structure. Consequently, the conformational plasticity of the protein is most likely related to its low overall stability rather than an intrinsically flexible protein structure. The well-defined structure supports a model of molecular recognition dominated by conformational selection, whereas only minor structural adjustments are necessary after the association.

The C-terminal tail of human neuronal calcium sensor 1 regulates the conformational stability of the Ca²âºâ‚‹ activated state.

Neuronal calcium sensor 1 (NCS-1) and orthologs are expressed in all organisms from yeast to humans. In the latter, NCS-1 plays an important role in neurotransmitter release and interacts with a plethora of binding partners mostly through a large solvent-exposed hydrophobic crevice. The structural basis behind the multispecific binding profile is not understood. To begin to address this, we applied NMR spectroscopy to determine the solution structure of calcium-bound human NCS-1. The structure in solution demonstrates interdomain flexibility and, in the absence of a binding partner, the C-terminal tail residues occupy the hydrophobic crevice as a ligand mimic. A variant with a C-terminal tail deletion shows lack of a defined structure but maintained cooperative unfolding and dramatically reduced global stability. The results suggest that the C-terminal tail is important for regulating the conformational stability of the Ca(2+)-activated state. Furthermore, a single amino acid mutation that was recently diagnosed in a patient with autistic spectrum disorder was seen to affect the C-terminal tail and binding crevice in NCS-1.

The interplay between transient α-helix formation and side chain rotamer distributions in disordered proteins probed by methyl chemical shifts.

The peptide backbones of disordered proteins are routinely characterized by NMR with respect to transient structure and dynamics. Little experimental information is, however, available about the side chain conformations and how structure in the backbone affects the side chains. Methyl chemical shifts can in principle report the conformations of aliphatic side chains in disordered proteins and in order to examine this two model systems were chosen: the acid denatured state of acyl-CoA binding protein (ACBP) and the intrinsically disordered activation domain of the activator for thyroid hormone and retinoid receptors (ACTR). We find that small differences in the methyl carbon chemical shifts due to the γ-gauche effect may provide information about the side chain rotamer distributions. However, the effects of neighboring residues on the methyl group chemical shifts obscure the direct observation of γ-gauche effect. To overcome this, we reference the chemical shifts to those in a more disordered state resulting in residue specific random coil chemical shifts. The (13)C secondary chemical shifts of the methyl groups of valine, leucine, and isoleucine show sequence specific effects, which allow a quantitative analysis of the ensemble of χ(2)-angles of especially leucine residues in disordered proteins. The changes in the rotamer distributions upon denaturation correlate to the changes upon helix induction by the co-solvent trifluoroethanol, suggesting that the side chain conformers are directly or indirectly related to formation of transient α-helices.

Sequence correction of random coil chemical shifts: correlation between neighbor correction factors and changes in the Ramachandran distribution.

Random coil chemical shifts are necessary for secondary chemical shift analysis, which is the main NMR method for identification of secondary structure in proteins. One of the largest challenges in the determination of random coil chemical shifts is accounting for the effect of neighboring residues. The contributions from the neighboring residues are typically removed by using neighbor correction factors determined based on each residue's effect on glycine chemical shifts. Due to its unusual conformational freedom, glycine may be particularly unrepresentative for the remaining residue types. In this study, we use random coil peptides containing glutamine instead of glycine to determine the random coil chemical shifts and the neighbor correction factors. The resulting correction factors correlate to changes in the populations of the major wells in the Ramachandran plot, which demonstrates that changes in the conformational ensemble are an important source of neighbor effects in disordered proteins. Glutamine derived random coil chemical shifts and correction factors modestly improve our ability to predict (13)C chemical shifts of intrinsically disordered proteins compared to existing datasets, and may thus improve the identification of small populations of transient structure in disordered proteins.

Random coil chemical shift for intrinsically disordered proteins: effects of temperature and pH.

Secondary chemical shift analysis is the main NMR method for detection of transiently formed secondary structure in intrinsically disordered proteins. The quality of the secondary chemical shifts is dependent on an appropriate choice of random coil chemical shifts. We report random coil chemical shifts and sequence correction factors determined for a GGXGG peptide series following the approach of Schwarzinger et al. (J Am Chem Soc 123(13):2970-2978, 2001). The chemical shifts are determined at neutral pH in order to match the conditions of most studies of intrinsically disordered proteins. Temperature has a non-negligible effect on the (13)C random coil chemical shifts, so temperature coefficients are reported for the random coil chemical shifts to allow extrapolation to other temperatures. The pH dependence of the histidine random coil chemical shifts is investigated in a titration series, which allows the accurate random coil chemical shifts to be obtained at any pH. By correcting the random coil chemical shifts for the effects of temperature and pH, systematic biases of the secondary chemical shifts are minimized, which will improve the reliability of detection of transient secondary structure in disordered proteins.

Cooperative formation of native-like tertiary contacts in the ensemble of unfolded states of a four-helix protein.

In studies of the ensembles of unfolded structures of a four-helix bundle protein, we have detected the presence of potential precursors of native tertiary structures. These observations were based on the perturbation of NMR chemical shifts of the protein backbone atoms by single site mutations. Some mutations change the chemical shifts of residues remote from the site of mutation indicating the presence of an interaction between the mutated and the remote residues, suggesting that the formation of helix segments and helix-helix interactions is cooperative. We can begin to track down the folding mechanism of this protein using only experimental data by combining the information available for the rate limiting structure formation during the folding process with measurements of the site specific hydrogen bond formation in the burst phase, and with the existence prior to the folding reaction of tertiary structures in the ensemble of otherwise unfolded structures observed in the present study.

Conformational selection in the molten globule state of the nuclear coactivator binding domain of CBP.

Native molten globules are the most folded kind of intrinsically disordered proteins. Little is known about the mechanism by which native molten globules bind to their cognate ligands to form fully folded complexes. The nuclear coactivator binding domain (NCBD) of CREB binding protein is particularly interesting in this respect as structural studies of its complexes have shown that NCBD folds into two remarkably different states depending on the ligand being ACTR or IRF-3. The ligand-free state of NCBD was characterized in order to understand the mechanism of folding upon ligand binding. Biophysical studies show that despite the molten globule nature of the domain, it contains a small cooperatively folded core. By NMR spectroscopy, we have demonstrated that the folded core of NCBD has a well ordered conformer with specific side chain packing. This conformer resembles the structure of the NCBD in complex with the protein ligand, ACTR, suggesting that ACTR binds to prefolded NCBD molecules from the ensemble of interconverting structures.

Temperature-dependent structural changes in intrinsically disordered proteins: formation of alpha-helices or loss of polyproline II?

Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient alpha-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations.

Residue-specific description of non-native transient structures in the ensemble of acid-denatured structures of the all-beta protein c-src SH3.

Secondary chemical shift analysis has been used to characterize the unfolded state of acid-denatured c-src SH3. Even though native c-src SH3 adopts an all-beta fold, we found evidence of transient helicity in regions corresponding to native loops. In particular, residues 40-46, connecting the n-src loop to the third beta-strand, exhibited an apparent helicity of nearly 45%. Furthermore, the RT loop and the diverging turn appeared to adopt non-native-like helical conformations. Interestingly, none of the residues found in transient helical conformations exhibited significant varphi-values [Riddle, D. S., et al. (1999) Nat. Struct. Biol. 6, 1016-1024]. This indicated that the transient helicity has no influence or only a weak influence on the actual protein folding reaction. The residual structural propensities were compared to those of other SH3 domains, revealing heterogeneity in the unfolded ensemble that clearly contrasts with the conserved character of the topology of native state and transition state ensembles typical for SH3 domains.