Identification and functional characterization of the bile acid transport proteins in non-mammalian ileum and mammalian liver

It has been proposed that intracellular carrier proteins mediate active transport of the bile acids within hepatocytes and ileocytes, during the enterohepatic circulation. In mammalian species only ileal bile acid binding proteins have been so far identified, while liver cytosolic carriers have never been found. On the contrary, in non-mammalian vertebrates, only liver, and not ileal, bile acid binding proteins were reported. The aim of the present work is to find the missing cytosolic transport proteins. A bioinformatic search allowed us to identify a non-mammalian putative bile acid binding protein in the chicken ileum (cI-BABP), which we recombinantly expressed and purified. The protein exhibits the capability, tested by in vitro NMR experiments, of binding bile acids. Furthermore, strong NMR evidence reported that the human liver fatty acid binding protein (hL-FABP) can also bind bile acids. Taken together, these data strongly suggest that both cI-BABP and hL-FABP have a bile acid binding function in the two organisms, and support a previous hypothesis on the role of hL-FABP in regulating bile acid metabolism and determining bile acid pool size.     

Solid-state NMR studies of the structure and mechanisms of proteins

Magic-angle spinning solid-state NMR experiments are well suited to investigating the structures and mechanisms of important proteins that are inaccessible to X-ray crystallography and solution NMR spectroscopy, including membrane proteins and disease-related protein aggregates. Good progress has been made in the development of methods for the complete structure determination of small (<20 kDa) solid proteins using uniformly 13C, 15N-labeled samples. Studies of selectively labeled proteins focusing on labeled active sites have yielded insights into the mechanisms of enzymes and of membrane proteins involved in energy and signal transduction. Studies of selectively labeled synthetic peptides have yielded structural models for biomedically important systems, including amyloid fibrils and surface-associated peptides involved in biomineralization and cell adhesion. Novel NMR and biochemical methods are being developed to target solid-state NMR experiments within large proteins and whole cells. These approaches are being used to investigate mechanisms of transmembrane signaling by membrane receptors and to characterize binding interactions between antibiotics and bacterial cell walls. Thus, solid-state NMR is proving to be a valuable biophysical tool for probing structure and dynamics in a wide range of biomolecules.     

Internal motions and exchange processes in human ileal bile acid binding protein as studied by backbone (15)N nuclear magnetic resonance spectroscopy

Human ileal bile acid binding protein (I-BABP), a member of the family of intracellular lipid binding proteins, is thought to play a role in the enterohepatic circulation of bile salts. Previously, we have shown by stopped-flow fluorescence analysis that positive binding cooperativity exhibited by I-BABP in its interactions with glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two primary bile salts in humans, is related to a slow conformational change in the protein. In this study, we used backbone (15)N relaxation nuclear magnetic resonance (NMR) techniques to obtain residue-specific information about the internal dynamics of apo I-BABP and the doubly ligated I-BABP:GCA:GCDA complex on various time scales. According to our NMR data, bile salt binding is accompanied by a slight rigidification of the (15)N-(1)H bond vectors on the picosecond to nanosecond time scale, with most pronounced changes occurring in the C-D region. In contrast to the minor effects of ligation on fast motions, relaxation dispersion NMR experiments indicate a marked difference between the two protein states on the microsecond to millisecond time scale. In the apo form, an extensive network of conformational fluctuations is detected throughout segments of the EFGHIJ β-strands and the C-D loop, which cease upon complexation. Our NMR data are in agreement with a conformational selection model we proposed earlier for I-BABP and support the hypothesis of an allosteric mechanism of ligand binding. According to the NMR measurements, the helical cap region may have a less crucial role in mediating ligand entry and release than what has been indicated for fatty acid binding proteins.     

Binding and folding of melittin in the presence of ganglioside GM1 micelles

In this work we examine the binding and folding of the membrane-active peptide, melittin in the presence of ganglioside GM1 micelle. The membrane bilayer is capable of inducing folding to small proteins and peptides upon binding. Using two-dimensional NMR techniques we have shown that at low concentration, GM1 micelle is able to induce an extended helical conformation to MLT. The pulsed-field gradient diffusion NMR study indicates that the peptide partition into GM1 micelle along with about 32% binding. While looking for the binding between MLT and GM1 using saturation transfer difference NMR spectroscopy, Val5, Leu9, Thr11, Ile17, Ser18, and Trp19 have been identified as the residues that are in close proximity to GM1 micelles.     

Recombinant pheromone binding protein 1 from Mamestra brassicae (MbraPBP1). Functional and structural characterization.

Pheromone binding proteins (PBPs) are small proteins (17 kDa on average) present at high concentrations ( approximately 10 mM) in the sensillum lymph of Lepidoptera antennae, where they play a key role in the perception of pheromones. By expression in Escherichia coli, we have obtained large quantities (2-3 mg.L-1) of pure, soluble, Mamestra brassicae PBP1 (MbraPBP1). These quantities are compatible with the requirements of X-ray and NMR studies. The recombinant protein has been characterized by native-polyacrylamide gel electrophoresis, Western blotting, N-terminal sequencing, mass spectrometry, gel filtration, circular dichroism, and NMR. Moreover, the recombinant MbraPBP1 has been shown to be able to bind the specific pheromone and a structural analogue, Z11-16:TFMK (cis-11-hexadecenyl trifluoromethyl ketone), in displacement experiments. Our results on MbraPBP1 confirm and extend previous findings on PBPs. MbraPBP1 and two PBPs from different species have been found to exist as dimers under nondenaturing conditions. The CD and structural prediction data confirm a markedly helical structure for insect PBPs rather than the beta-barrel fold found in vertebrates odorant binding proteins. We have tentatively identified the location of the helices and the short beta-strands with respect to the binding site. Currently we have obtained small diffracting crystals of the recombinant MbraPBP1 and determined their space group and molecular content.     

Protein-ligand interactions measured by 15N-filtered diffusion experiments

NMR diffusion coefficient measurements have been shown to be sensitive to the conformational and oligomeric states of proteins. Recently, heteronuclear-filtered diffusion experiments have been proposed [Dingley et al. (1997) J. Biomol. NMR, 10, 1–8]. Several new heteronuclear-filtered diffusion pulse sequences are proposed which are shown to have superior sensitivity to those previously proposed. One of these new heteronuclear-filtered diffusion experiments has been used to study the binding of an SH3 domain to a peptide. Using this system, we show that it is possible to measure binding constants from diffusion coefficient measurements.     

NMR spectroscopy in structure-based drug design

NMR methods for the study of motion in proteins continue to improve, and a number of studies of protein-ligand complexes relevant to drug design have been reported over the past year, for example, studies of fatty-acid-binding protein and SH2 and SH3 domains. These studies have begun to give a picture of the structural dynamics of protein-ligand complexes and to relate the changes in dynamics on ligand binding to the origins of specificity. NMR is also valuable in locating binding sites, both qualitatively from changes in chemical shift and more precisely from distances measured from relaxation effects. The conformation of the bound ligand can provide useful information for drug design, and over the past year improvements in methods have made it easier to obtain quantitative information from transferred nuclear Overhauser effect experiments.     

Strategies for the uses of lanthanide NMR shift probes in the determination of protein structure in solutio. Application to the EF calcium binding site of carp parvalbumin.

The homologous sequences observed for many calcium binding proteins such as parvalbumin, troponin C, the myosin light chains, and calmodulin has lead to the hypothesis that these proteins have homologous structures at the level of their calcium binding sites. This paper discusses the development of a nuclear magnetic resonance (NMR) technique which will enable us to test this structural hypothesis in solution. The technique involves the substitution of a paramagnetic lanthanide ion for the calcium ion which results in lanthanide induced shifts and broadening in the 1H NMR spectrum of the protein. These shifts are sensitive monitors of the precise geometrical orientation of each proton nucleus relative to the metal. The values of several parameters in the equation relating the NMR shifts to the structure are however known as priori. We have attempted to determine these parameters, the orientation and principal elements of the magnetic susceptibility tensor of the protein bound metal, by studying the lanthanide induced shifts for the protein parvalbumin whose structure has been determined by x-ray crystallographic techniques. The interaction of the lanthanide ytterbium with parvalbumin results in high resolution NMR spectra exhibiting a series of resonances with shifts spread over the range 32 to -19 ppm. The orientation and principal elements of the ytterbium magnetic susceptibility tensor have been determined using three assigned NMR resonances, the His-26 C2 and C4 protons and the amino terminal acetyl protons, and seven methyl groups; all with known geometry relative to the EF calcium binding site. The elucidation of these parameters has allowed us to compare the observed spectrum of the nuclei surrounding the EF calcium binding site of parvalbumin with that calculated from the x-ray structure. A significant number of the calculated shifts are larger than any of the observed shifts. We feel that a refinement of the x-ray based proton coordinates will be possible utilizing the geometric information contained in the lanthanide shifted NMR spectrum.     

Backbone 1H, 15N, 13C and Ile, Leu, Val methyl chemical shift assignments for the 33.5 kDa N-terminal domain of Candida albicans ALS1

The agglutinin-like-sequence (ALS) family of adhesion proteins are a key virulence factor for C. albicans. These proteins have been implicated in several functions, notably adhesion and invasion of different cell types, as well as binding to peptides and proteins in the cell surface and extracellular matrix. In order to understand their binding mechanism and en route to a full structural determination by NMR, here we report the resonance assignments of backbone atoms plus Ile, Leu and Val methyls for residues 18–329 of ALS1, which comprises the 33.5 kDa binding domain.     

Detection and characterization of xenon-binding sites in proteins by 129Xe NMR spectroscopy

Xenon-binding sites in proteins have led to a number of applications of xenon in biochemical and structural studies. Here we further develop the utility of 129Xe NMR in characterizing specific xenon-protein interactions. The sensitivity of the 129Xe chemical shift to its local environment and the intense signals attainable by optical pumping make xenon a useful NMR reporter of its own interactions with proteins. A method for detecting specific xenon-binding interactions by analysis of 129Xe chemical shift data is illustrated using the maltose binding protein (MBP) from Escherichia coli as an example. The crystal structure of MBP in the presence of 8atm of xenon confirms the binding site determined from NMR data. Changes in the structure of the xenon-binding cavity upon the binding of maltose by the protein can account for the sensitivity of the 129Xe chemical shift to MBP conformation. 129Xe NMR data for xenon in solution with a number of cavity containing phage T4 lysozyme mutants show that xenon can report on cavity structure. In particular, a correlation exists between cavity size and the binding-induced 129Xe chemical shift. Further applications of 129Xe NMR to biochemical assays, including the screening of proteins for xenon binding for crystallography are considered.     

RNA binding strategies of ribosomal proteins.

Structures of a number of ribosomal proteins have now been determined by crystallography and NMR, though the complete structure of a ribosomal protein-rRNA complex has yet to be solved. However, some ribosomal protein structures show strong similarity to well-known families of DNA or RNA binding proteins for which structures in complex with cognate nucleic acids are available. Comparison of the known nucleic acid binding mechanisms of these non-ribosomal proteins with the most highly conserved surfaces of similar ribosomal proteins suggests ways in which the ribosomal proteins may be binding RNA. Three binding motifs, found in four ribosomal proteins so far, are considered here: homeodomain-like alpha-helical proteins (L11), OB fold proteins (S1 and S17) and RNP consensus proteins (S6). These comparisons suggest that ribosomal proteins combine a small number of fundamental strategies to develop highly specific RNA recognition sites.     

Intrinsically unstructured domain 3 of hepatitis C Virus NS5A forms a "fuzzy complex" with VAPB-MSP domain which carries ALS-causing mutations

Hepatitis C virus (HCV) affects nearly 200 million people worldwide and is a leading factor for serious chronic liver diseases. For replicating HCV genome, the membrane-associated replication machinery needs to be formed by both HCV non-structural proteins including NS5A and human host factors. Recently NS5A has been identified to bind ER-anchored human VAP proteins and consequently this interaction may serve as a novel target for design of anti-HCV drugs. So far no biophysical characterization of this interaction has been reported. Here, we dissected the 243-residue VAPB into 4 and 447-residue NS5A into 10 fragments, followed by CD and NMR characterization of their structural properties. Subsequently, binding interactions between these fragments have been extensively assessed by NMR HSQC titration which is very powerful in detecting even very weak binding. The studies lead to three important findings: 1). a "fuzzy complex" is formed between the intrinsically-unstructured third domain (D3) of NS5A and the well-structured MSP domain of VAPB, with an average dissociation constant (Kd) of ~5 μM. 2). The binding-important residues on both NS5A-D3 and VAPB-MSP have been successfully mapped out, which provided experimental constraints for constructing the complex structure. In the complex, unstructured D3 binds to three surface pockets on one side of the MSP structure. Interestingly, two ALS-causing mutations T46I and P56S are also located on the D3-MSP interface. Moreover, NS5A-D3, FFAT-containing proteins and EphA4 appear to have overlapped binding interfaces on the MSP domain. 3). NS5A-D3 has been experimentally confirmed to competes with EphA4 in binding to the MSP domain, and T46I mutation of MSP dramatically abolishes its binding ability to D3. Our study not only provides essential foundation for further deciphering structure and function of the HCV replication machinery, but may also shed light on rationalizing a recent observation that a chronic HCV patient surprisingly developed ALS-like syndrome.     

An NMR analysis of the binding of inhibitors to yeast phosphoglycerate kinase

The binding of a series of inhibitors to the enzyme phosphoglycerate kinase has been studied using NMR to uncover the binding sites and the effects of binding on the protein conformation. The very effective inhibitor, Suramin, causes the most pronounced changes. The design of inhibitors for mobile proteins is discussed.     

A 31P MAS NMR study of cytidine 2'-phosphate bound to ribonuclease A in the crystalline state

31P CP/MAS spectra have been obtained from 2'-CMP bound to ribonuclease A in the crystalline state. The chemical shift value is closely similar to that found in solution NMR studies under similar conditions, and corresponds to that of the dianionic state of the free compound. It is suggested that the NMR approach may be of general applicability for the comparison of the binding properties of small molecules to proteins in crystals and solution.     

Solution structure of the single-stranded DNA binding protein of the filamentous Pseudomonas phage Pf3: similarity to other proteins binding to single-stranded nucleic acids.

The three-dimensional structure of the homodimeric single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been determined using heteronuclear multidimensional NMR techniques and restrained molecular dynamics. NMR experiments and structure calculations have been performed on a mutant protein (Phe36 --> His) that was successfully designed to reduce the tendency of the protein to aggregate. The protein monomer is composed of a five-stranded antiparallel beta-sheet from which two beta-hairpins and a large loop protrude. The structure is compared with the single-stranded DNA binding protein encoded by the filamentous Escherichia coli phage Ff, a protein with a similar biological function and DNA binding properties, yet quite different amino acid sequence, and with the major cold shock protein of Escherichia coli, a single-stranded DNA binding protein with an entirely different sequence, biological function and binding characteristics. The amino acid sequence of the latter is highly homologous to the nucleic acid binding domain (i.e. the cold shock domain) of proteins belonging to the Y-box family. Despite their differences in amino acid sequence and function, the folds of the three proteins are remarkably similar, suggesting that this is a preferred folding pattern shared by many single-stranded DNA binding proteins.     

Insulin-like growth factor binding protein-2: NMR analysis and structural characterization of the N-terminal domain

The insulin-like growth factor binding proteins are a family of six proteins (IGFBP-1 to -6) that bind insulin-like growth factors-I and -II (IGF-I/II) with high affinity. In addition to regulating IGF actions, IGFBPs have IGF-independent functions. IGFBP-2, the largest member of this family, is over-expressed in many cancers and has been proposed as a possible target for the development of novel anti-cancer therapeutics. The IGFBPs have a common architecture consisting of conserved N- and C-terminal domains joined by a variable linker domain. The solution structure and dynamics of the C-terminal domain of human IGFBP-2 have been reported (Kuang Z. et al. J. Mol. Biol. 364, 690-704, 2006) but neither the N-domain (N-BP-2) nor the linker domain have been characterised. Here we present NMR resonance assignments for human N-BP-2, achieved by recording spectra at low protein concentration using non-uniform sampling and maximum entropy reconstruction. Analysis of secondary chemical shifts shows that N-BP-2 possesses a secondary structure similar to that of other IGFBPs. Although aggregation hampered determination of the solution structure for N-BP-2, a homology model was generated based on the high degree of sequence and structure homology exhibited by the IGFBPs. This model was consistent with experimental NMR and SAXS data and displayed some unique features such as a Pro/Ala-rich non-polar insert, which formed a flexible solvent-exposed loop on the surface of the protein opposite to the IGF-binding interface. NMR data indicated that this loop could adopt either of two alternate conformations in solution - an entirely flexible conformation and one containing nascent helical structure. This loop and an adjacent poly-proline sequence may comprise a potential SH3 domain interaction site for binding to other proteins.     

Characterization of a novel interaction between Bcl-2 members Diva and Harakiri

Interactions within proteins of the Bcl-2 family are key in the regulation of apoptosis. The death-inducing members control apoptotic mechanisms partly by antagonizing the prosurvival proteins through heterodimer formation. Structural and biophysical studies on these complexes are providing important clues to understand their function. To help improve our knowledge on protein-protein interactions within the Bcl-2 family we have studied the binding between two of its members: mouse Diva and human Harakiri. Diva has been shown to perform both prosurvival and killing activity. In contrast, Harakiri induces cell death by interacting with antiapoptotic Bcl-2 members. Here we show using ELISA and NMR that Diva and Harakiri can interact in vitro. Combining the NMR data with the previously reported three-dimensional structure of Diva we find that Harakiri binds to a specific region in Diva. This interacting surface is equivalent to the known binding area of prosurvival Bcl-2 members from the reported structures of the complexes, suggesting that Diva could function at the structural level similarly to the antiapoptotic proteins of the Bcl-2 family. We illustrate this result by building a structural model of the heterodimer using molecular docking and the NMR data as restraints. Moreover, combining circular dichroism and NMR we also show that Harakiri is largely unstructured with residual (13%) α-helical conformation. This result agrees with intrinsic disorder previously observed in other Bcl-2 members. In addition, Harakiri constructs of different length were studied to identify the region critical for the interaction. Differential affinity for Diva of these constructs suggests that the amino acid sequence flanking the interacting region could play an important role in binding.     

Properties of thrombin- and elastase-modified human antithrombin III

Proteolytically modified forms of human antithrombin III have been prepared by reaction of native antithrombin with thrombin, human neutrophil elastase, or porcine pancreatic elastase. These forms have two chains disulfide linked and are of the same molecular weight as native antithrombin III. 1H NMR spectroscopy has been used to characterize these proteins and to compare them to one another and to native antithrombin III. The three modified proteins have very similar NMR spectra and histidine residues with identical pH titration parameters, and they undergo the same spectral changes upon binding heparin. They differ from native antithrombin III in all of these respects. In addition, the proteins are much more stable than native antithrombin III. The three modified proteins behave identically as a function of temperature; at 372 K, 44 K above the unfolding temperature for native antithrombin III, the proteins are still folded and possess approximately 70 unexchanged amide protons even after several hours. The unfolding of the heparin binding domain at low concentrations of deuteriated guanidine hydrochloride seen in native thrombin III is absent in the modified forms. It is concluded that the thrombin- and elastase-modified forms of antithrombin have identical structures when allowance is made for the slightly different sites of cleavage by the two types of elastase and by thrombin. This structure is very different from that of native antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)