NMR Studies of the Interactions between Sialyllactoses and the Polysialytransferase Domain for Polysialylation Inhibition.

It is known that sialyllactose (SL) in mammalians is a major source of sialic acid (Sia), which can further form cytidine monophosphate sialic acid (CMP-Sia), and the final product is polysialic acid (polySia) using polysialyltransferases (polySTs) on the neural cell adhesion molecule (NCAM). This process is called NCAM polysialylation. The overexpression of polysialylation is strongly related to cancer cell migration, invasion, and metastasis. In order to inhibit the overexpression of polysialylation, in this study, SL was selected as an inhibitor to test whether polysialylation could be inhibited. Our results suggest that the interactions between the polysialyltransferase domain (PSTD) in polyST and CMP-Siaand the PSTD and polySia could be inhibited when the 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL) concentration is about 0.5 mM or 6'-SL and 3 mM, respectively. The results also show that SLs (particularly for 3'-SL) are the ideal inhibitors compared with another two inhibitors, low-molecular-weight heparin (LMWH) and cytidine monophosphate (CMP), because 3'-SL can not only be used to inhibit NCAM polysialylation, but is also one of the best supplements for infant formula and the gut health system.

Molecular Interactions of the Polysialytransferase Domain (PSTD) in ST8Sia IV with CMP-Sialic Acid and Polysialic Acid Required for Polysialylation of the Neural Cell Adhesion Molecule Proteins: An NMR Study.

Polysialic acid (polySia) is an unusual glycan that posttranslational modifies neural cell adhesion molecule (NCAM) proteins in mammalian cells. The up-regulated expression of polySia-NCAM is associated with tumor progression in many metastatic human cancers and in neurocognitive processes. Two members of the ST8Sia family of α2,8-polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST) both catalyze synthesis of polySia when activated cytidine monophosphate(CMP)-Sialic acid (CMP-Sia) is translocate into the lumen of the Golgi apparatus. Two key polybasic domains in the polySTs, the polybasic region (PBR) and the polysialyltransferase domain (PSTD) areessential forpolysialylation of the NCAM proteins. However, the precise molecular details to describe the interactions required for polysialylation remain unknown. In this study, we hypothesize that PSTD interacts with both CMP-Sia and polySia to catalyze polysialylation of the NCAM proteins. To test this hypothesis, we synthesized a 35-amino acid-PSTD peptide derived from the ST8Sia IV gene sequence and used it to study its interaction with CMP-Sia, and polySia. Our results showed for the PSTD-CMP-Sia interaction,the largest chemical-shift perturbations (CSP) were in amino acid residues V251 to A254 in the short H1 helix, located near the N-terminus of PSTD. However, larger CSP values for the PSTD-polySia interaction were observed in amino acid residues R259 to T270 in the long H2 helix. These differences suggest that CMP-Sia preferentially binds to the domain between the short H1 helix and the longer H2 helix. In contrast, polySia was principally bound to the long H2 helix of PSTD. For the PSTD-polySia interaction, a significant decrease in peak intensity was observed in the 20 amino acid residues located between the N-and C-termini of the long H2 helix in PSTD, suggesting a slower motion in these residues when polySia bound to PSTD. Specific features of the interactions between PSTD-CMP-Sia, and PSTD-polySia were further confirmed by comparing their 800 MHz-derived HSQC spectra with that of PSTD-Sia, PSTD-TriSia (DP 3) and PSTD-polySia. Based on the interactions between PSTD-CMP-Sia, PSTD-polySia, PBR-NCAM and PSTD-PBR, these findingsprovide a greater understanding of the molecular mechanisms underlying polySia-NCAM polysialylation, and thus provides a new perspective for translational pharmacological applications and development by targeting the two polysialyltransferases.

Conformational switch of the bacterial adhesin FimH in the absence of the regulatory domain: Engineering a minimalistic allosteric system.

For many biological processes such as ligand binding, enzymatic catalysis, or protein folding, allosteric regulation of protein conformation and dynamics is fundamentally important. One example is the bacterial adhesin FimH, where the C-terminal pilin domain exerts negative allosteric control over binding of the N-terminal lectin domain to mannosylated ligands on host cells. When the lectin and pilin domains are separated under shear stress, the FimH-ligand interaction switches in a so-called catch-bond mechanism from the low- to high-affinity state. So far, it has been assumed that the pilin domain is essential for the allosteric propagation within the lectin domain that would otherwise be conformationally rigid. To test this hypothesis, we generated mutants of the isolated FimH lectin domain and characterized their thermodynamic, kinetic, and structural properties using isothermal titration calorimetry, surface plasmon resonance, nuclear magnetic resonance, and X-ray techniques. Intriguingly, some of the mutants mimicked the conformational and kinetic behaviors of the full-length protein and, even in absence of the pilin domain, conducted the cross-talk between allosteric sites and the mannoside-binding pocket. Thus, these mutants represent a minimalistic allosteric system of FimH, useful for further mechanistic studies and antagonist design.

Parvulin 17-catalyzed Tubulin Polymerization Is Regulated by Calmodulin in a Calcium-dependent Manner.

Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTP-dependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca(2+)-loaded calmodulin (Ca(2+)/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca(2+)/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly. In contrast, parvulin 14 (Par14), which lacks only the first 25 N-terminal residues of the Par17 sequence, does not interact with Ca(2+)/CaM, indicating that this interaction is exclusive for Par17. Pulldown experiments and chemical shift perturbation analysis with (15)N-labeled Par17 furthermore confirmed that calmodulin (CaM) interacts in a Ca(2+)-dependent manner with the Par17 N terminus. The reverse experiment with (15)N-labeled Ca(2+)/CaM demonstrated that the N-terminal Par17 segment binds to both CaM lobes simultaneously, indicating that Ca(2+)/CaM undergoes a conformational change to form a binding channel between its two lobes, apparently similar to the structure of the CaM-smMLCK(796-815) complex. In vitro tubulin polymerization assays furthermore showed that Ca(2+)/CaM completely suppresses Par17-promoted microtubule assembly. The results imply that Ca(2+)/CaM binding to the N-terminal segment of Par17 causes steric hindrance of the Par17 active site, thus interfering with the Par17/tubulin interaction. This Ca(2+)/CaM-mediated control of Par17-assisted microtubule assembly may provide a mechanism that couples Ca(2+) signaling with microtubule function.

Insights into the binding of Ag ions with SilE model peptides: an NMR and MS coupled approach.

The diffuse and renewed use of silver as antimicrobial agent has caused the development of resistance to silver ions in some bacterial strains, posing a serious threat for health systems. In order to cast light on the mechanistic features of resistance, here, we aimed to understand how silver interacts with the periplasmic metal-binding protein SilE which is engaged in bacterial silver detoxification. This aim was addressed by studying two peptide portions of SilE sequence (SP2 and SP3) that contain the putative motifs involved in Ag+ binding. We demonstrate that SP2 model peptide is involved in silver binding through its histidine and methionine residues in the two HXXM binding sites. In particular, the first binding site is supposed to bind the Ag+ ion in a linear fashion, while the second binding site complexes the silver ion in a distorted trigonal planar fashion. We propose a model where the SP2 peptide binds two silver ions when the concentration ratio Ag+/SP2 is ≥10.0. We also suggest that the two binding sites of SP2 have different affinity for silver. This evidence comes from the change in the path direction of the Nuclear Magnetic Resonance (NMR) cross-peaks upon the addition of Ag+. Here, we report the conformational changes of SilE model peptides occurring upon silver binding, monitored at a deep level of molecular details. This was addressed by a multifaceted approach, combining NMR, circular dichroism, and mass spectrometry experiments.

BCL-2 Protein Family Interaction Analysis by Nuclear Magnetic Resonance Spectroscopy.

Biomolecular nuclear magnetic resonance (NMR) is a powerful and versatile method for studying both protein-protein interactions (PPIs) and protein-small molecule binding. NMR has been used extensively in the investigation of BCL-2 family proteins revealing the structure of key family members, identifying binding partners and interaction sites, and screening small molecule modulators. In this chapter we discuss the application of NMR to identify interaction sites and structure determination of protein-protein and protein-small molecule complexes using two examples.

NMR Spectroscopy to Study MAP Kinase Binding to MAP Kinase Phosphatases.

NMR spectroscopy and other solution methods are increasingly being used to obtain novel insights into the mechanisms by which MAPK regulatory proteins bind and direct the activity of MAPKs. Here, we describe how interactions between the MAPK p38α and its regulatory proteins are studied using NMR spectroscopy, isothermal titration calorimetry, and small angle X-ray scattering (SAXS).