Ligand-modulated parallel mechanical unfolding pathways of maltose-binding proteins

Protein folding and unfolding are complex phenomena, and it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) is a large (a two-domain, 370-amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of MBP and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) whereas the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events, and the remaining 38% follow path II. In the case of maltose-bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) with that of maltose. The precursor protein preMBP also shows a similar behavior upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively, for preMBP. These observations demonstrate that ligand binding can modulate the mechanical unfolding pathways of proteins by a kinetic partitioning mechanism. This could be a general mechanism in the unfolding of other large two-domain ligand-binding proteins of the bacterial periplasmic space.     

Nanomechanics of the cadherin ectodomain: "canalization" by Ca2+ binding results in a new mechanical element

Cadherins form a large family of calcium-dependent cell-cell adhesion receptors involved in development, morphogenesis, synaptogenesis, differentiation, and carcinogenesis through signal mechanotransduction using an adaptor complex that connects them to the cytoskeleton. However, the molecular mechanisms underlying mechanotransduction through cadherins remain unknown, although their extracellular region (ectodomain) is thought to be critical in this process. By single molecule force spectroscopy, molecular dynamics simulations, and protein engineering, here we have directly examined the nanomechanics of the C-cadherin ectodomain and found it to be strongly dependent on the calcium concentration. In the presence of calcium, the ectodomain extends through a defined ("canalized") pathway that involves two mechanical resistance elements: a mechanical clamp from the cadherin domains and a novel mechanostable component from the interdomain calcium-binding regions ("calcium rivet") that is abolished by magnesium replacement and in a mutant intended to impede calcium coordination. By contrast, in the absence of calcium, the mechanical response of the ectodomain becomes largely "decanalized" and destabilized. The cadherin ectodomain may therefore behave as a calcium-switched "mechanical antenna" with very different mechanical responses depending on calcium concentration (which would affect its mechanical integrity and force transmission capability). The versatile mechanical design of the cadherin ectodomain and its dependence on extracellular calcium facilitate a variety of mechanical responses that, we hypothesize, could influence the various adhesive properties mediated by cadherins in tissue morphogenesis, synaptic plasticity, and disease. Our work represents the first step toward the mechanical characterization of the cadherin system, opening the door to understanding the mechanical bases of its mechanotransduction.     

Effect of Src kinase phosphorylation on disordered C-terminal domain of N-methyl-D-aspartic acid (NMDA) receptor subunit GluN2B protein

NMDA receptors are ligand-gated ion channels with a regulatory intracellular C-terminal domain (CTD). In GluN2B, the CTD is the largest domain in the protein but is intrinsically disordered. The GluN2B subunit is the major tyrosine-phosphorylated protein in synapses. Src kinase phosphorylates the GluN2B CTD, but it is unknown how this affects channel activity. In disordered proteins, phosphorylation can tip the balance between order and disorder. Transitions can occur in both directions, so it is not currently possible to predict the effects of phosphorylation. We used single molecule fluorescence to characterize the effects of Src phosphorylation on GluN2B. Scanning fluorescent labeling sites throughout the domain showed no positional dependence of the energy transfer. Instead, efficiency only scaled with the separation between labeling sites suggestive of a relatively featureless conformational energy landscape. Src phosphorylation led to a general expansion of the polypeptide, which would result in greater exposure of known protein-binding sites and increase the physical separation between contiguous sites. Phosphorylation makes the CTD more like a random coil leaving open the question of how Src exerts its effects on the NMDA receptor.     

Constitutive activation of nuclear factor-E2-related factor 2 induces biotransformation enzyme and transporter expression in livers of mice with hepatocyte-specific deletion of Kelch-like ECH-associated protein 1

Chemicals that activate nuclear factor-E2-related factor 2 (Nrf2) often increase multidrug-resistance-associated protein (Mrp) expression in liver. Hepatocyte-specific deletion of Kelch-like ECH-associated protein 1 (Keap1) activates Nrf2. Use of hepatocyte-specific Keap1 deletion represents a nonpharmacological method to determine whether constitutive Nrf2 activation upregulates liver transporter expression in vivo. The mRNA, protein expression, and localization of several biotransformation and transporters were determined in livers of wild-type and hepatocyte-specific Keap1-null mice. Sulfotransferase 2a1/2, NADP(H):quinone oxidoreductase 1, cytochrome P450 2b10, 3a11, and glutamate-cysteine ligase catalytic subunit expression were increased in livers of Keap1-null mice. Organic anion-transporting polypeptide 1a1 expression was nearly abolished, as compared to that detected in livers of wild-type mice. By contrast, Mrp 1-5 mRNA and protein levels were increased in Keap1-null mouse livers, with Mrp4 expression being more than 15-fold higher than wild types. In summary, Nrf2 has a significant role in affecting Oatp and Mrp expressions.     

Src homology 2 domains of protein tyrosine phosphatase are associated in vitro with both the insulin receptor and insulin receptor substrate-1 via different phosphotyrosine motifs

To clarify the role of protein tyrosine phosphatase containing Src homology 2 (SH2) regions on insulin signaling, we investigated the interactions among the insulin receptor, a pair of SH2 domains of SH-PTP2 coupled to glutathione-S-transferase (GST) and insulin receptor substrate-1 (IRS-1)-GST fusion proteins (amino-portion, IRS-1N; carboxyl portion, IRS-1C). GST-SH2 protein of SH-PTP2 bound to the wild type insulin receptor, but not to that with a carboxyl-terminal mutation (Y/F2). Furthermore, even though Y/F2 receptors were used, the SH2 protein was also co-immunoprecipitated with IRS-1C, but not with IRS-1N. These results indicate that SH2 domains of SH-PTP2 can directly associate with the Y¹³²²TXM motif on the carboxyl terminus of insulin receptors and also may bind to the carboxyl portion of IRS-1, possibly via the V¹¹⁷²IDL motif in vitro.     

External Ba2+ block of the two-pore domain potassium channel TREK-1 defines conformational transition in its selectivity filter

TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba(2+) blocked the TREK-1 channel in a concentration- and time-dependent manner. A mutagenesis analysis showed that overlapped binding of Ba(2+) at the assumed K(+) binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba(2+) was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K(+)-free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (ΔN41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K(+) and proton.     

Solution structure of the phosphotyrosine binding (PTB) domain of human tensin2 protein in complex with deleted in liver cancer 1 (DLC1) peptide reveals a novel peptide binding mode

The protein deleted in liver cancer 1 (DLC1) interacts with the tensin family of focal adhesion proteins to play a role as a tumor suppressor in a wide spectrum of human cancers. This interaction has been proven to be crucial to the oncogenic inhibitory capacity and focal adhesion localization of DLC1. The phosphotyrosine binding (PTB) domain of tensin2 predominantly interacts with a novel site on DLC1, not the canonical NPXY motif. In this study, we characterized this interaction biochemically and determined the complex structure of tensin2 PTB domain with DLC1 peptide by NMR spectroscopy. Our HADDOCK-derived complex structure model elucidates the molecular mechanism by which tensin2 PTB domain recognizes DLC1 peptide and reveals a PTB-peptide binding mode that is unique in that peptide occupies the binding site opposite to the canonical NPXY motif interaction site with the peptide utilizing a non-canonical binding motif to bind in an extended conformation and that the N-terminal helix, which is unique to some Shc- and Dab-like PTB domains, is required for binding. Mutations of crucial residues defined for the PTB-DLC1 interaction affected the co-localization of DLC1 and tensin2 in cells and abolished DLC1-mediated growth suppression of hepatocellular carcinoma cells. This tensin2 PTB-DLC1 peptide complex with a novel binding mode extends the versatile binding repertoire of the PTB domains in mediating diverse cellular signaling pathways as well as provides a molecular and structural basis for better understanding the tumor-suppressive activity of DLC1 and tensin2.