IGF-dependent dynamic modulation of a protease cleavage site in the intrinsically disordered linker domain of human IGFBP2.

Functional regulation via conformational dynamics is well known in structured proteins but less well characterized in intrinsically disordered proteins and their complexes. Using NMR spectroscopy, we have identified a dynamic regulatory mechanism in the human insulin-like growth factor (IGF) system involving the central, intrinsically disordered linker domain of human IGF-binding protein-2 (hIGFBP2). The bioavailability of IGFs is regulated by the proteolysis of IGF-binding proteins. In the case of hIGFBP2, the linker domain (L-hIGFBP2) retains its intrinsic disorder upon binding IGF-1, but its dynamics are significantly altered, both in the IGF binding region and distantly located protease cleavage sites. The increase in flexibility of the linker domain upon IGF-1 binding may explain the IGF-dependent modulation of proteolysis of IGFBP2 in this domain. As IGF homeostasis is important for cell growth and function, and its dysregulation is a key contributor to several cancers, our findings open up new avenues for the design of IGFBP analogs inhibiting IGF-dependent tumors.

Cooperativity of membrane-protein and protein-protein interactions control membrane remodeling by epsin 1 and affects clathrin-mediated endocytosis.

Membrane remodeling is a critical process for many membrane trafficking events, including clathrin-mediated endocytosis. Several molecular mechanisms for protein-induced membrane curvature have been described in some detail. Contrary, the effect that the physico-chemical properties of the membrane have on these processes is far less well understood. Here, we show that the membrane binding and curvature-inducing ENTH domain of epsin1 is regulated by phosphatidylserine (PS). ENTH binds to membranes in a PI(4,5)P2-dependent manner but only induces curvature in the presence of PS. On PS-containing membranes, the ENTH domain forms rigid homo-oligomers and assembles into clusters. Membrane binding and membrane remodeling can be separated by structure-to-function mutants. Such oligomerization mutants bind to membranes but do not show membrane remodeling activity. In vivo, they are not able to rescue defects in epidermal growth factor receptor (EGFR) endocytosis in epsin knock-down cells. Together, these data show that the membrane lipid composition is important for the regulation of protein-dependent membrane deformation during clathrin-mediated endocytosis.

Membrane-embedded TSPO: an NMR view.

Translocator Protein (18 kDa) (TSPO) is a mitochondrial transmembrane protein commonly used as a biomarker for neuroinflammation and is also a potential therapeutic target in neurodegenerative diseases. Despite intensive research efforts, the function of TSPO is still largely enigmatic. Deciphering TSPO structure in the native lipid environment is essential to gain insight into its cellular activities and to design improved diagnostic and therapeutic ligands. Here, we discuss the influence of lipid composition on the structure of mammalian TSPO embedded into lipid bilayers on the basis of solid-state NMR experiments. We further highlight that cholesterol can influence both the tertiary and quaternary TSPO structure and also influence TSPO localization in mitochondria-associated endoplasmic reticulum membranes.

Challenges and approaches to understand cholesterol-binding impact on membrane protein function: an NMR view.

Experimental evidence for a direct role of lipids in determining the structure, dynamics, and function of membrane proteins leads to the term 'functional lipids'. In particular, the sterol molecule cholesterol modulates the activity of many membrane proteins. The precise nature of cholesterol-binding sites and the consequences of modulation of local membrane micro-viscosity by cholesterol, however, is often unknown. Here, we review the current knowledge of the interaction of cholesterol with transmembrane proteins, with a special focus on structural aspects of the interaction derived from nuclear magnetic resonance approaches. We highlight examples of the importance of cholesterol modulation of membrane protein function, discuss the specificity of cholesterol binding, and review the proposed binding motifs from a molecular perspective. We conclude with a short perspective on what could be future trends in research efforts targeted towards a better understanding of cholesterol/membrane protein interactions.

Insights into Cholesterol/Membrane Protein Interactions Using Paramagnetic Solid-State NMR.

Cholesterol is an essential component of animal cell membranes and impacts the structure and function of membrane proteins. But how cholesterol exerts its functions remains often enigmatic. Here, high-resolution solid-state NMR in combination with paramagnetic cholesterol analogues was shown to be a powerful approach to study the interaction of membrane proteins with cholesterol. Application of the method to the 169-residue translocator protein TSPO provides residue-specific information about its interaction with cholesterol. Comparison with NMR signal perturbations induced by diamagnetic cholesterol furthermore supports changes in the structure of mammalian TSPO caused by cholesterol binding.

Rapid NMR Assignments of Proteins by Using Optimized Combinatorial Selective Unlabeling.

A new approach for rapid resonance assignments in proteins based on amino acid selective unlabeling is presented. The method involves choosing a set of multiple amino acid types for selective unlabeling and identifying specific tripeptides surrounding the labeled residues from specific 2D NMR spectra in a combinatorial manner. The methodology directly yields sequence specific assignments, without requiring a contiguously stretch of amino acid residues to be linked, and is applicable to deuterated proteins. We show that a 2D [(15) N,(1) H] HSQC spectrum with two 2D spectra can result in ∼50 % assignments. The methodology was applied to two proteins: an intrinsically disordered protein (12 kDa) and the 29 kDa (268 residue) α-subunit of Escherichia coli tryptophan synthase, which presents a challenging case with spectral overlaps and missing peaks. The method can augment existing approaches and will be useful for applications such as identifying active-site residues involved in ligand binding, phosphorylation, or protein-protein interactions, even prior to complete resonance assignments.

Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti-apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain-transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein-ligand interactions, we have determined the sequence-specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple-resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2-aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd.

Structure of the mammalian TSPO/PBR protein.

The 3D structure of the 18-kDa transmembrane (TM) protein TSPO (translocator protein)/PBR (peripheral benzodiazepine receptor), which contains a binding site for benzodiazepines, is important to better understand its function and regulation by endogenous and synthetic ligands. We have recently determined the structure of mammalian TSPO/PBR in complex with the diagnostic ligand PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; Jaremko et al. (2014) Science 343: , 1363-1366], providing for the first time atomic-level insight into the conformation of this protein, which is up-regulated in various pathological conditions including Alzheimer's disease and Parkinson's disease. Here, we review the studies which have probed the structural properties of mammalian TSPO/PBR as well as the homologues bacterial tryptophan-rich sensory proteins (TspOs) over the years and provide detailed insight into the 3D structure of mouse TSPO (mTSPO)/PBR in complex with PK11195.

A fast NMR method for resonance assignments: application to metabolomics.

We present a new method for rapid NMR data acquisition and assignments applicable to unlabeled ((12)C) or (13)C-labeled biomolecules/organic molecules in general and metabolomics in particular. The method involves the acquisition of three two dimensional (2D) NMR spectra simultaneously using a dual receiver system. The three spectra, namely: (1) G-matrix Fourier transform (GFT) (3,2)D [(13)C, (1)H] HSQC-TOCSY, (2) 2D (1)H-(1)H TOCSY and (3) 2D (13)C-(1)H HETCOR are acquired in a single experiment and provide mutually complementary information to completely assign individual metabolites in a mixture. The GFT (3,2)D [(13)C, (1)H] HSQC-TOCSY provides 3D correlations in a reduced dimensionality manner facilitating high resolution and unambiguous assignments. The experiments were applied for complete (1)H and (13)C assignments of a mixture of 21 unlabeled metabolites corresponding to a medium used in assisted reproductive technology. Taken together, the experiments provide time gain of order of magnitudes compared to the conventional data acquisition methods and can be combined with other fast NMR techniques such as non-uniform sampling and covariance spectroscopy. This provides new avenues for using multiple receivers and projection NMR techniques for high-throughput approaches in metabolomics.

Efficient sequential assignments in proteins with reduced dimensionality 3D HN(CA)NH.

We present reduced dimensionality (RD) 3D HN(CA)NH for efficient sequential assignment in proteins. The experiment correlates the (15)N and (1)H chemical shift of a residue ('i') with those of its immediate N-terminal (i - 1) and C-terminal (i + 1) neighbors and provides four-dimensional chemical shift correlations rapidly with high resolution. An assignment strategy is presented which combines the correlations observed in this experiment with amino acid type information obtained from 3D CBCA(CO)NH. By classifying the 20 amino acid types into seven distinct categories based on (13)C(ß) chemical shifts, it is observed that a stretch of five sequentially connected residues is sufficient to map uniquely on to the polypeptide for sequence specific resonance assignments. This method is exemplified by application to three different systems: maltose binding protein (42 kDa), intrinsically disordered domain of insulin-like growth factor binding protein-2 and Ubiquitin. Fast data acquisition is demonstrated using longitudinal (1)H relaxation optimization. Overall, 3D HN(CA)NH is a powerful tool for high throughput resonance assignment, in particular for unfolded or intrinsically disordered polypeptides.

High resolution methyl selective ¹³C-NMR of proteins in solution and solid state.

New ¹³C-detected NMR experiments have been devised for molecules in solution and solid state, which provide chemical shift correlations of methyl groups with high resolution, selectivity and sensitivity. The experiments achieve selective methyl detection by exploiting the one bond J-coupling between the ¹³C-methyl nucleus and its directly attached ¹³C spin in a molecule. In proteins such correlations edit the ¹³C-resonances of different methyl containing residues into distinct spectral regions yielding a high resolution spectrum. This has a range of applications as exemplified for different systems such as large proteins, intrinsically disordered polypeptides and proteins with a paramagnetic centre.

GFT projection NMR for efficient (1)H/ (13)C sugar spin system identification in nucleic acids.

A newly implemented G-matrix Fourier transform (GFT) (4,3)D HC(C)CH experiment is presented in conjunction with (4,3)D HCCH to efficiently identify (1)H/(13)C sugar spin systems in (13)C labeled nucleic acids. This experiment enables rapid collection of highly resolved relay 4D HC(C)CH spectral information, that is, shift correlations of (13)C-(1)H groups separated by two carbon bonds. For RNA, (4,3)D HC(C)CH takes advantage of the comparably favorable 1'- and 3'-CH signal dispersion for complete spin system identification including 5'-CH. The (4,3)D HC(C)CH/HCCH based strategy is exemplified for the 30-nucleotide 3'-untranslated region of the pre-mRNA of human U1A protein.

Amino acid selective labeling and unlabeling for protein resonance assignments.

Structural characterization of proteins by NMR spectroscopy begins with the process of sequence specific resonance assignments in which the (1)H, (13)C and (15)N chemical shifts of all backbone and side-chain nuclei in the polypeptide are assigned. This process requires different isotope labeled forms of the protein together with specific experiments for establishing the sequential connectivity between the neighboring amino acid residues. In the case of spectral overlap, it is useful to identify spin systems corresponding to the different amino acid types selectively. With isotope labeling this can be achieved in two ways: (i) amino acid selective labeling or (ii) amino acid selective 'unlabeling'. This chapter describes both these methods with more emphasis on selective unlabeling describing the various practical aspects. The recent developments involving combinatorial selective labeling and unlabeling are also discussed.

Amino acid selective unlabeling for sequence specific resonance assignments in proteins.

Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective 'unlabeling' or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly (13)C/(15)N labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {(12)CO( i )-(15)N( i+1)}-filtered HSQC, which aids in linking the (1)H(N)/(15)N resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i - 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to (2)H labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of (14)N at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies.

High-resolution methyl edited GFT NMR experiments for protein resonance assignments and structure determination.

Three-dimensional (3D) structure determination of proteins is benefitted by long-range distance constraints comprising the methyl groups, which constitute the hydrophobic core of proteins. However, in methyl groups (of Ala, Ile, Leu, Met, Thr and Val) there is a significant overlap of ¹³C and ¹H chemical shifts. Such overlap can be resolved using the recently proposed (3,2)D HCCH-COSY, a G-matrix Fourier transform (GFT) NMR based experiment, which facilitates editing of methyl groups into distinct spectral regions by combining their ¹³C chemical shifts with that of the neighboring, directly attached, ¹³C nucleus. Using this principle, we present three GFT experiments: (a) (4,3)D NOESY-HCCH, (b) (4,3)D ¹H-TOCSY-HCCH and (c) (4,3)D ¹³C-TOCSY-HCCH. These experiments provide unique 4D spectral information rapidly with high sensitivity and resolution for side-chain resonance assignments and NOE analysis of methyl groups. This is exemplified by (4,3)D NOESY-HCCH data acquired for 17.9 kDa non-deuterated cytosolic human J-protein co-chaperone, which provided crucial long-range distance constraints for its 3D structure determination.

Solid-state NMR investigation of the involvement of the P2 region in tau amyloid fibrils.

The aggregation of hyperphosphorylated tau into amyloid fibrils is closely linked to the progression of Alzheimer's disease. To gain insight into the link between amyloid structure and disease, the three-dimensional structure of tau fibrils has been studied using solid-state NMR (ssNMR) and cryogenic electron microscopy (cryo-EM). The proline-rich region of tau remains poorly defined in the context of tau amyloid structures, despite the clustering of several phosphorylation sites, which have been associated with Alzheimer's disease. In order to gain insight into the contribution of the proline-rich region P2 of tau to amyloid fibrils, we studied in vitro aggregated amyloid fibrils of tau constructs, which contain both the proline-rich region P2 and the pseudo-repeats. Using ssNMR we show that the sequence [Formula: see text], the most hydrophobic patch within the P2 region, loses its flexibility upon formation of amyloid fibrils. The data suggest a contribution of the P2 region to tau amyloid fibril formation, which might account for some of the unassigned electron density in cryo-EM studies of tau fibrils and could be modulated by tau phosphorylation at the disease-associated AT180 epitope T231/S235.

Cholesterol-mediated allosteric regulation of the mitochondrial translocator protein structure.

Cholesterol is an important regulator of membrane protein function. However, the exact mechanisms involved in this process are still not fully understood. Here we study how the tertiary and quaternary structure of the mitochondrial translocator protein TSPO, which binds cholesterol with nanomolar affinity, is affected by this sterol. Residue-specific analysis of TSPO by solid-state NMR spectroscopy reveals a dynamic monomer-dimer equilibrium of TSPO in the membrane. Binding of cholesterol to TSPO's cholesterol-recognition motif leads to structural changes across the protein that shifts the dynamic equilibrium towards the translocator monomer. Consistent with an allosteric mechanism, a mutation within the oligomerization interface perturbs transmembrane regions located up to 35 Å away from the interface, reaching TSPO's cholesterol-binding motif. The lower structural stability of the intervening transmembrane regions provides a mechanistic basis for signal transmission. Our study thus reveals an allosteric signal pathway that connects membrane protein tertiary and quaternary structure with cholesterol binding.

Structure and Characterisation of a Key Epitope in the Conserved C-Terminal Domain of the Malaria Vaccine Candidate MSP2.

Merozoite surface protein 2 (MSP2) is an intrinsically disordered antigen that is abundant on the surface of the malaria parasite Plasmodium falciparum. The two allelic families of MSP2, 3D7 and FC27, differ in their central variable regions, which are flanked by highly conserved C-terminal and N-terminal regions. In a vaccine trial, full-length 3D7 MSP2 induced a strain-specific protective immune response despite the detectable presence of conserved region antibodies. This work focuses on the conserved C-terminal region of MSP2, which includes the only disulphide bond in the protein and encompasses key epitopes recognised by the mouse monoclonal antibodies 4D11 and 9H4. Although the 4D11 and 9H4 epitopes are overlapping, immunofluorescence assays have shown that the mouse monoclonal antibody 4D11 binds to MSP2 on the merozoite surface with a much stronger signal than 9H4. Understanding the structural basis for this antigenic difference between these antibodies will help direct the design of a broad-spectrum and MSP2-based malaria vaccine. 4D11 and 9H4 were reengineered into antibody fragments [variable region fragment (Fv) and single-chain Fv (scFv)] and were validated as suitable models for their full-sized IgG counterparts by surface plasmon resonance and isothermal titration calorimetry. An alanine scan of the 13-residue epitope 3D7-MSP2207-222 identified the minimal binding epitope of 4D11 and the key residues involved in binding. A 2.2-Å crystal structure of 4D11 Fv bound to the eight-residue epitope NKENCGAA provided valuable insight into the possible conformation of the C-terminal region of MSP2 on the parasite. This work underpins continued efforts to optimise recombinant MSP2 constructs for evaluation as potential vaccine candidates.

Crowding interactions perturb structure and stability by destabilizing the stable core of the α-subunit of tryptophan synthase.

The consequences of crowding derived from relatively small and intrinsically disordered proteins are not clear yet. We report the effect of ficoll-70 on the structure and stability of native and partially folded states of the 29 kDa alpha subunit of tryptophan synthase (αTS). Overall, combining the changes in the circular dichroism and fluorescence spectra, in conjunction with the gradual loss of cooperativity under urea denaturation in the presence of increasing amounts of ficoll, it may be concluded that the crowding agent perturbs not only the native state but also the partially folded state of αTS. Importantly, NMR data indicate that ficoll interacts with the residues that constitute the stable core of the protein thus shedding light on the origin of the observed perturbation.