NMR structural biology of sulfated glycans

Sulfated fucans, sulfated galactans, and glycosaminoglycans are extensively studied worldwide in terms of both structure and biomedical functions. Liquid-state nuclear magnetic resonance (NMR) spectroscopy is the most employed analytical technique in structural analysis of these sulfated glycans. This is due to the fact that NMR-based analyses enable a series of achievements such as (i) accurate structure characterization/determination; (ii) measurements of parameters regarding molecular motion (dynamics); (iii) assessment of the 3D structures (usually assisted by computational techniques of Molecular Modeling and/or Molecular Dynamics) of the composing monosaccharides (ring conformers) and the overall conformational states of the glycan chains either free in solution or bound to proteins; and (iv) analysis of the resultant intermolecular complexes with functional proteins through either the protein or the carbohydrate perspective. In this review, after a general introduction about the principal NMR parameters utilized for achieving this set of structural information, discussion is given on NMR-based studies of some representative sulfated fucans, sulfated galactans, and glycosaminoglycans. Due to the growing number of studies concerning both structure and function of sulfated glycans and the widely use of NMR spectroscopy in such studies, a review paper discussing (i) the most experiments employed for analysis, (ii) procedures used in data interpretation, and (iii) the general aspects of the sulfated glycans, is timely in the literature.     

Downhill versus Barrier-Limited Folding of BBL: 3. Heterogeneity of the Native State of the BBL Peripheral Subunit Binding Domain and Its Implications for Folding Mechanisms

Protein folding studies are generally predicated on Anfinsen's dogma that there is a unique native state of a protein. However, this is not always the case. NMR measurements of BBL, for example, find a decrease in helicity of helix 2 surrounding His166 on its protonation, which, with other experimental data, suggests that the native state can occupy two or more conformations. Here, we analysed the native structure of BBL as a function of pH, temperature and ionic strength, along with a truncated BBL construct, by extensive all-atom molecular dynamics simulations in explicit solvent, corresponding to at least 400 ns of trajectories collected for each set of conditions. The native state was heterogeneous under a variety of conditions, consisting of two predominant conformations. This equilibrium changed with conditions: protonation of His166 at low pH shifted the equilibrium in favour of a less ordered conformer, while high ionic strength at neutral pH shifted the equilibrium to a more ordered conformer. Furthermore, high temperature and truncation of the sequence also shifted the equilibrium toward the less ordered conformer. Importantly, conformational heterogeneity in a native structure that changes with conditions will lead to deviations from the classic two-state behaviour during the barrier-limited unfolding of a protein. In particular, some regions of the protein will appear to unfold asynchronously and some residues will have anomalous thermal titration curves and unusual baseline behaviour monitored microscopically by NMR spectroscopy and macroscopically by calorimetry and other techniques. Such data could otherwise be interpreted as evidence for barrier-free downhill folding. Any biological significance of downhill folding of BBL appears to be ruled out by recent crystallographic studies on the reaction cycle of the BBL-equivalent domain in a pyruvate dehydrogenase multienzyme complex in which the domain remains of constant structure.     

Structure of the Third Intracellular Loop of the Vasopressin V2 Receptor and Conformational Changes upon Binding to gC1qR

The V2 vasopressin receptor is a G-protein-coupled receptor that regulates the renal antidiuretic response. Its third intracellular loop is involved in the coupling not only with the GαS protein but also with gC1qR, a potential chaperone of G-protein-coupled receptors. In this report, we describe the NMR solution structure of the V2 i3 loop under a cyclized form (i3_cyc) and characterize its interaction with gC1qR. i3_cyc formed a left-twisted α-helical hairpin structure. The building of a model of the entire V2 receptor including the i3_cyc NMR structure clarified the side-chain orientation of charged residues, in agreement with literature mutagenesis reports. In the model, the i3 loop formed a rigid helical column, protruding deep inside the cytoplasm, as does the i3 loop in the recently elucidated structure of squid rhodopsin. However, its higher packing angle resulted in a different structural motif at the intracellular interface, which may be important for the specific recognition of GαS. Moreover, we could estimate the apparent K_d of the i3_cyc/gC1qR complex by anisotropy fluorescence. Using a shorter and more soluble version of i3_cyc, which encompassed the putative site of gC1qR binding, we showed by NMR saturation transfer difference spectroscopy that the binding surface corresponded to the central arginine cluster. Binding to gC1qR induced the folding of the otherwise disordered short peptide into a spiral-like path formed by a succession of I and IV turns. Our simulations suggested that this folding would rigidify the arginine cluster in the entire i3 loop and would alter the conformation of the cytosolic extensions of TM V and TM VI helices. In agreement with this conformational rearrangement, we observed that binding of gC1qR to the full-length receptor modifies the intrinsic tryptophan fluorescence binding curves of V2 to an antagonist.     

Atypical binding of the Swa2p UBA domain to ubiquitin

Swa2p is an auxilin-like yeast protein that is involved in vesicular transport and required for uncoating of clathrin-coated vesicles. Swa2p contains a ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin (Ub)-mediated processes. We have determined a structural model of the Swa2p UBA domain in complex with Ub using NMR spectroscopy and molecular docking. Ub recognition occurs predominantly through an atypical interaction in which UBA helix α1 and the N-terminal part of helix α2 bind to Ub. Mutation of Ala148, a key residue in helix α1, to polar residues greatly reduced the affinity of the UBA domain for Ub and revealed a second low-affinity Ub-binding site located on the surface formed by helices α1 and α3. Surface plasmon resonance showed that the Swa2p UBA domain binds K48- and K63-linked di-Ub in a non-linkage-specific manner. These results reveal convergent evolution of a Ub-binding site on helix α1 of UBA domains involved in membrane protein trafficking.     

Fas Apoptosis Inhibitory Molecule Contains a Novel β-Sandwich in Contact with a Partially Ordered Domain

Fas apoptosis inhibitory molecule (FAIM) is a soluble cytosolic protein inhibitor of programmed cell death and is found in organisms throughout the animal kingdom. A short isoform of FAIM is expressed in all tissue types, while an alternatively spliced long isoform is specifically expressed in the brain. Here, the short isoform is shown to consist of two independently folding domains in contact with each other. The NMR solution structure of the C-terminal domain of murine FAIM is solved in isolation and revealed to be a novel protein fold, a noninterleaved seven-stranded β-sandwich. The structure and sequence reveal several residues that are likely to be involved in functionally significant interactions with the N-terminal domain or other binding partners. Chemical shift perturbation is used to elucidate contacts made between the N-terminal domain and the C-terminal domain.     

PILZ Protein Structure and Interactions with PILB and the FIMX EAL Domain: Implications for Control of Type IV Pilus Biogenesis

The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since then, a large and diverse family of bacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not PilZ itself, have been shown to bind the important bacterial second messenger bis(3′→5′)cyclic diGMP (c-diGMP). The crystal structures of the PilZ orthologs from Xanthomonas axonopodis pv citri (PilZ_XAC1133, this work) and from Xanthomonas campestris pv campestris (XC1028) present significant structural differences to other PilZ homologs that explain its failure to bind c-diGMP. NMR analysis of PilZ_XAC1133 shows that these structural differences are maintained in solution. In spite of their emerging importance in bacterial signaling, the means by which PilZ proteins regulate specific processes is not clear. In this study, we show that PilZ_XAC1133 binds to PilB, an ATPase required for T4P polymerization, and to the EAL domain of FimX_XAC2398, which regulates T4P biogenesis and localization in other bacterial species. These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed between any PilZ domain and a target protein. While we were unable to detect phosphodiesterase activity for FimX_XAC2398in vitro, we show that it binds c-diGMP both in the presence and in the absence of PilZ_XAC1133. Site-directed mutagenesis studies for conserved and exposed residues suggest that PilZ_XAC1133 interactions with FimX_XAC2398 and PilB_XAC3239 are mediated through a hydrophobic surface and an unstructured C-terminal extension conserved only in PilZ orthologs. The FimX-PilZ-PilB interactions involve a full set of “degenerate” GGDEF, EAL and PilZ domains and provide the first evidence of the means by which PilZ orthologs and FimX interact directly with the TP4 machinery.     

Chelerythrine and sanguinarine dock at distinct sites on BclXL that are not the classic BH3 binding cleft

The ratio of the levels of pro-survival and pro-apoptotic members of the Bcl-2 protein family is thought to be an important regulatory factor for determining the sensitivity of the mammalian cells to apoptotic stimuli. High levels of expression of pro-survival members such as Bcl(XL) in human cancers were frequently found to be a good prognostic indicator predicting poor response to chemotherapy. The pro-survival members of the Bcl-2 family mediate their effects through heterodimerization with the BH3 region of the pro-apoptotic members. Structural analyses of the binding complex of the BH3 peptide and Bcl(XL) showed that a hydrophobic groove termed the BH3 binding cleft is the docking site for the BH3 region. Chemical mimetics of the BH3 region such as BH3I-1 that target the BH3 binding cleft indeed exhibit pro-apoptotic activities. Chelerythrine (CHE) and sanguinarine (SAN) are natural benzophenanthridine alkaloids that are structurally homologous to each other. CHE was previously identified as an inhibitor of Bcl(XL) function from a high-throughput screen of natural products, but its mode of interaction with Bcl(XL) is not known. By determining the effect of site-directed mutagenesis on ligand binding and using saturation transfer difference (STD) NMR experiments, we have verified locations of these docked ligands. Surprisingly, CHE and SAN bind separately at the BH groove and BH1 region of Bcl(XL) respectively, different from the BH3 binding cleft where other known inhibitors of Bcl(XL) target. Interestingly, certain residues on the flexible loop between helices alpha1 and alpha2 of Bcl(XL) are also perturbed upon CHE, but not SAN or BH3I-1 binding. Although CHE and SAN are similarly effective as BH3I-1 in displacing bound BH3 peptide, they are much more effective in inducing apoptosis, raising the possibility that CHE and SAN might be able to antagonize other pro-survival mechanisms in addition to the one that involves BH3 region binding.