New approaches to structure determination by NMR spectroscopy.

New strategies have recently been developed for studying biological macromolecules of large size (beyond 100 kDa) in order to both improve the quality of the structures and make structure determination more efficient. This has been achieved by utilizing cross-correlation effects and novel labeling strategies, and developing novel NMR spectroscopy experiments.

Methods for Studying Interactions Between Atg8/LC3/GABARAP and LIR-Containing Proteins.

LC3/GABARAP proteins (LC3/GABARAPs) are mammalian orthologues of yeast Atg8, small ubiquitin (Ub)-like proteins (UBLs) whose covalent attachment to lipid membranes is crucial for the growth and closure of the double membrane vesicle called the autophagosome. In the past decade, it was demonstrated that Atg8/LC3/GABARAPs are also required for autophagic degradation of cargos in a selective fashion. Cargo selectivity is ensured by receptor proteins, such as p62/SQSTM1, NBR1, Cue5, Atg19, NIX, Atg32, NCOA4, and FAM134B, which simultaneously bind Atg8/LC3/GABARAPs and the cargo together, thereby linking the core autophagic machinery to the target structure: a protein, an organelle, or a pathogen. LC3-interacting regions (LIRs) are short linear motifs within selective autophagy receptors and some other structural and signaling proteins (e.g., ULK1, ATG13, FIP200, and Dvl2), which mediate binding to Atg8/LC3/GABARAPs. Identification and characterization of LIR-containing proteins have provided important insights into the biology of the autophagy pathway, and studying their interactions with the core autophagy machinery represents a growing area of autophagy research. Here, we present protocols for the identification of LIR-containing proteins, i.e., by yeast-two-hybrid screening, glutathione S-transferase (GST) pulldown experiments, and peptide arrays. The use of two-dimensional peptide arrays also represents a powerful method to identify the residues of the LIR motif that are critical for binding. We also describe a biophysical method for studying interactions between Atg8/LC3/GABARAP and LIR-containing proteins and a protocol for preparation and purification of LIR peptides.

The role of protein-solvent interactions in protein unfolding.

Protein unfolding occurs when the balance of forces between the protein's interaction with itself and the protein's interaction with its environment is disrupted. The disruption of this balance of forces may be as simple as a perturbance of the normal water structure around the protein. A decrease in the normal water-water interaction will result in an increase in the relative interaction of water with the protein. An increase in the number of interactions between water and the protein may initiate a protein's unfolding. This model for protein unfolding is supported by a range of recent experimental and computational data.

Functional interplay between MDM2, p63/p73 and mutant p53.

Many cancers express mutant p53 proteins that have lost wild-type tumor suppressor activity and, in many cases, have acquired oncogenic functions that can contribute to tumor progression. These activities of mutant p53 reflect interactions with several other proteins, including the p53 family members p63 and p73. Mutations in p53 that affect protein conformation (such as R175H) show strong binding to p63 and p73, whereas p53 mutants that only mildly affect the conformation (such as R273H) bind less well. A previously described aggregation domain of mutant p53 is not required for p63 or p73 binding; indeed, mutations within this region lead to the acquisition of a mutant p53 phenotype-including a conformational shift, p63/p73 binding and the ability to promote invasion. The activity of wild-type p53 is regulated by an interaction with MDM2 and we have investigated the potential role of MDM2 in the mutant p53/p63/p73 interactions. Both mutant p53 and p73 bind MDM2 well, whereas p63 binds much more weakly. We found that MDM2 can inhibit p63 binding to p53R175H but enhances the weaker p53R273H/p73 interaction. These effects on the interactions are reflected in an ability of MDM2 to relieve the inhibition of p63 by p53R175H, but enhance the inhibition of p73 activity by p53R175H and R273H. We propose a model in which MDM2 competes with p63 for binding to p53R175H to restore p63 activity, but forms a trimeric complex with p73 and p53R273H to more strongly inhibit p73 function.

Interaction of urea with an unfolded protein. The DNA-binding domain of the 434-repressor.

Experimental techniques are presented for the observation of the solvation of the unfolded form of a globular protein, the N-terminal 63-residue polypeptide from the 434 repressor, in 7 M aqueous urea solution by both water and urea. With the use of 15N-labelled urea it is demonstrated that the cross sections through two-dimensional nuclear Overhauser enhancement (NOE) spectra at the chemical shifts of H2O and urea both contain direct NOEs with the protein, under conditions where exchange peaks are observed only in the water cross section. A preliminary analysis of the data showed that the residence times of urea molecules in solvation sites near the methyl groups of Val, Leu and Ile are significantly longer than those of water molecules in the same sites.

Salt-stabilized globular protein structure in 7 M aqueous urea solution.

A 7 M aqueous urea solution of the 63-residue N-terminal domain of the 434-repressor at pH 7.5 and 18 degrees C contains a mixture of about 10% native, folded protein and 90% unfolded protein. Interconversion between the two conformations is slow on the NMR chemical shift time scale, so that observation of separate resonances can be used to monitor the equilibrium between folded and unfolded protein when changing the solution conditions. In this paper we describe the influence of various salts or non-ionic compounds on this conformational equilibrium. Solution conditions are described which contain a homogenous preparation of the folded protein in the presence of 6 to 7 M urea, providing a basis for an NMR structure determination in concentrated urea and for studies of the solvation of the folded protein in mixed water/urea/salt environments.

Analysis of the oligomeric state and transactivation potential of TAp73α.

The proteins p73 and p63 are members of the p53 protein family and are involved in important developmental processes. Their high sequence identity with the tumor suppressor p53 has suggested that they act as tumor suppressors as well. While p63 has a crucial role in the maintenance of epithelial stem cells and in the quality control of oocytes without a clear role as a tumor suppressor, p73's tumor suppressor activity is well documented. In a recent study we have shown that the transcriptional activity of TAp63α, the isoform responsible for the quality control in oocytes, is regulated by its oligomeric state. The protein forms an inactive, dimeric and compact conformation in resting oocytes, while the detection of DNA damage leads to the formation of an active, tetrameric and open conformation. p73 shows a high sequence identity to p63, including those domains that are crucial in stabilizing its inactive state, thus suggesting that p73's activity might be regulated by its oligomeric state as well. Here, we have investigated the oligomeric state of TAp73α by size exclusion chromatography and detailed domain interaction mapping, and show that in contrast to p63, TAp73α is a constitutive open tetramer. However, its transactivation potential depends on the cellular background and the promoter context. These results imply that the regulation of p73's transcriptional activity might be more closely related to p53 than to p63.

p63 and p73, the ancestors of p53.

p73 and p63 are two homologs of the tumor suppressive transcription factor p53. Given the high degree of structural similarity shared by the p53 family members, p73 and p63 can bind and activate transcription from the majority of the p53-responsive promoters. Besides overlapping functions shared with p53 (i.e., induction of apoptosis in response to cellular stress), the existence of extensive structural variability within the family determines unique roles for p63 and p73. Their crucial and specific functions in controlling development and differentiation are well exemplified by the p63 and p73 knockout mouse phenotypes. Here, we describe the contribution of p63 and p73 to human pathology with emphasis on their roles in tumorigenesis and development.

The C-terminus of p63 contains multiple regulatory elements with different functions.

The transcription factor p63 is expressed as at least six different isoforms, of which two have been assigned critical biological roles within ectodermal development and skin stem cell biology on the one hand and supervision of the genetic stability of oocytes on the other hand. These two isoforms contain a C-terminal inhibitory domain that negatively regulates their transcriptional activity. This inhibitory domain contains two individual components: one that uses an internal binding mechanism to interact with and mask the transactivation domain and one that is based on sumoylation. We have carried out an extensive alanine scanning study to identify critical regions within the inhibitory domain. These experiments show that a stretch of ∼13 amino acids is crucial for the binding function. Further, investigation of transcriptional activity and the intracellular level of mutants that cannot be sumoylated suggests that sumoylation reduces the concentration of p63. We therefore propose that the inhibitory function of the C-terminal domain is in part due to direct inhibition of the transcriptional activity of the protein and in part due to indirect inhibition by controlling the concentration of p63.

Conformational stability and activity of p73 require a second helix in the tetramerization domain.

p73 and p63, the two ancestral members of the p53 family, are involved in neurogenesis, epithelial stem cell maintenance and quality control of female germ cells. The highly conserved oligomerization domain (OD) of tumor suppressor p53 is essential for its biological functions, and its structure was believed to be the prototype for all three proteins. However, we report that the ODs of p73 and p63 differ from the OD of p53 by containing an additional alpha-helix that is not present in the structure of the p53 OD. Deletion of this helix causes a dissociation of the OD into dimers; it also causes conformational instability and reduces the transcriptional activity of p73. Moreover, we show that ODs of p73 and p63 strongly interact and that a large number of different heterotetramers are supported by the additional helix. Detailed analysis shows that the heterotetramer consisting of two homodimers is thermodynamically more stable than the two homotetramers. No heterooligomerization between p53 and the p73/p63 subfamily was observed, supporting the notion of functional orthogonality within the p53 family.

Large-scale production of functional membrane proteins.

The preparation of sufficient amounts of high-quality samples is still the major bottleneck for the characterization of membrane proteins by in vitro approaches. The hydrophobic nature, the requirement for complicated transport and modification pathways, and the often observed negative effects on membrane properties are intrinsic features of membrane proteins that frequently cause significant problems in overexpression studies. Establishing efficient protocols for the production of functionally folded membrane proteins is therefore a challenging task, and numerous specific characteristics have to be considered. In addition, a variety of expression systems have been developed, and choice of appropriate techniques could strongly depend on the desired target membrane proteins as well as on their intended applications. The production of membrane proteins is a highly dynamic field and new or modified approaches are frequently emerging. The review will give an overview of currently established processes for the production of functionally folded membrane proteins.

Ubiquitin binding mediates the NF-kappaB inhibitory potential of ABIN proteins.

Deregulated nuclear factor kappaB (NF-kappaB) activation plays an important role in inflammation and tumorigenesis. ABIN proteins have been characterized as negative regulators of NF-kappaB signaling. However, their mechanism of NF-kappaB inhibition remained unclear. With the help of a yeast two-hybrid screen, we identified ABIN proteins as novel ubiquitin-interacting proteins. The minimal ubiquitin-binding domain (UBD) corresponds to the ABIN homology domain 2 (AHD2) and is highly conserved in ABIN-1, ABIN-2 and ABIN-3. Moreover, this region is also present in NF-kappaB essential modulator/IkappaB kinase gamma (NEMO/IKKgamma) and the NEMO-like protein optineurin, and is therefore termed UBD in ABIN proteins and NEMO (UBAN). Nuclear magnetic resonance studies of the UBAN domain identify it as a novel type of UBD, with the binding surface on ubiquitin being significantly different from the binding surface of other UBDs. ABIN-1 specifically binds ubiquitinated NEMO via a bipartite interaction involving its UBAN and NEMO-binding domain. Mutations in the UBAN domain led to a loss of ubiquitin binding and impaired the NF-kappaB inhibitory potential of ABINs. Taken together, these data illustrate an important role for ubiquitin binding in the negative regulation of NF-kappaB signaling by ABINs and identify UBAN as a novel UBD.

NMR in the SPINE Structural Proteomics project.

This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless (13)C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations.

In-cell NMR spectroscopy.

The recent development of "in-cell NMR" techniques by two independent groups has demonstrated that NMR spectroscopy can be used to characterize the conformation and dynamics of biological macromolecules inside living cells. In this article, we describe different methods and discuss current and future applications as well as critical parameters of this new technique. We show experimental results, compare them with traditional in vitro experiments, and demonstrate that differences between the in vitro and the in vivo state of a macromolecule exist and can be detected and characterized.

Carbon-detected NMR experiments to investigate structure and dynamics of biological macromolecules.

We have started to develop new NMR pulse sequences that detect carbon magnetization during the acquisition period. These experiments have become possible with the recent introduction of cryogenic probe heads. We show that a careful design of these carbon-detected experiments can at least partially compensate for the inherent lower sensitivity of carbon detection compared to proton detection. We discuss potential applications of carbon detection and demonstrate a deconvolution technique that removes the effects of carbon-carbon couplings from the spectra.

Evaluation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectroscopy.

Our recently developed in-cell NMR procedure now enables one to observe protein conformations inside living cells. Optimization of the technique demonstrates that distinguishing the signals produced by a single protein species depends critically on protein overexpression levels and the correlation time in the cytoplasm. Less relevant is the selective incorporation of (15)N. Poorly expressed proteins, insoluble proteins, and proteins that cannot tumble freely due to associations within the cell cannot yet be observed. We show in-cell NMR spectra of bacterial NmerA and human calmodulin and discuss limitations of the technique as well as prospects for future applications.

An activation switch in the ligand binding pocket of the C5a receptor.

Although agonists are thought to occupy binding pockets within the seven-helix core of serpentine receptors, the topography of these binding pockets and the conformational changes responsible for receptor activation are poorly understood. To identify the ligand binding pocket in the receptor for complement factor 5a (C5aR), we assessed binding affinities of hexapeptide ligands, each mutated at a single position, for seven mutant C5aRs, each mutated at a single position in the putative ligand binding site. In ChaW (an antagonist) and W5Cha (an agonist), the side chains at position 5 are tryptophan and cyclohexylalanine, respectively. Comparisons of binding affinities indicated that the hexapeptide residue at this position interacts with two C5aR residues, Ile-116 (helix III) and Val-286 (helix VII); in a C5aR model these two side chains point toward one another. Both the I116A and the V286A mutations markedly increased binding affinity of W5Cha but not that of ChaW. Moreover, ChaW, the antagonist hexapeptide, acted as a full agonist on the I116A mutant. These results argue that C5aR residues Ile-116 and Val-286 interact with the side chain at position 5 of the hexapeptide ligand to form an activation switch. Based on this and previous work, we present a docking model for the hexapeptide within the C5aR binding pocket. We propose that agonists induce a small change in the relative orientations of helices III and VII and that these helices work together to allow movement of helix VI away from the receptor core, thereby triggering G protein activation.

Exploring the role of the solvent in the denaturation of a protein: a molecular dynamics study of the DNA binding domain of the 434 repressor.

Molecular dynamics simulations of the DNA binding domain of 434 repressor are presented which aim at unraveling the role of solvent in protein denaturation. Four altered solvent models, each mimicking various possible aspects of the addition of a denaturant to the aqueous solvent, were used in the simulations to analyze their effects on the stability of the protein. The solvent was altered by selectively changing the Coulombic interaction between water and protein atoms and between different water molecules. The use of a modified solvent model has the advantage of mimicking the presence of denaturant without having denaturant molecules present in the simulation, which would require much longer simulations. In these simulations, only an increase in the solvent-protein Coulombic interaction causes initiation of protein unfolding in a manner consistent with NMR data. The altered solvent thus provides a model of a denaturing environment for studying protein unfolding.

Solution structure of the core NFATC1/DNA complex.

The nuclear factor of the activated T cell (NFAT) family of transcription factors regulates cytokine gene expression by binding to the promoter/enhancer regions of antigen-responsive genes, usually in cooperation with heterologous DNA-binding partners. Here we report the solution structure of the binary complex formed between the core DNA-binding domain of human NFATC1 and the ARRE2 DNA site from the interleukin-2 promoter. The structure reveals that DNA binding induces the folding of key structural elements that are required for both sequence-specific recognition and the establishment of cooperative protein-protein contacts. The orientation of the NFAT DNA-binding domain observed in the binary NFATC1-DBD*/ DNA complex is distinct from that seen in the ternary NFATC2/AP-1/DNA complex, suggesting that the domain reorients upon formation of a cooperative transcriptional complex.