Kai-En Chen

Low-resolution solution structures of Munc18:Syntaxin protein complexes indicate an open binding mode driven by the Syntaxin N-peptide.

When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode.

The 1.2 A resolution crystal structure of TcpG, the Vibrio cholerae DsbA disulfide-forming protein required for pilus and cholera-toxin production

The enzyme TcpG is a periplasmic protein produced by the Gram-negative pathogen Vibrio cholerae. TcpG is essential for the production of ToxR-regulated proteins, including virulence-factor pilus proteins and cholera toxin, and is therefore a target for the development of a new class of anti-virulence drugs. Here, the 1.2 Å resolution crystal structure of TcpG is reported using a cryocooled crystal. This structure is compared with a previous crystal structure determined at 2.1 Å resolution from data measured at room temperature. The new crystal structure is the first DsbA crystal structure to be solved at a sufficiently high resolution to allow the inclusion of refined H atoms in the model. The redox properties of TcpG are also reported, allowing comparison of its oxidoreductase activity with those of other DSB proteins. One of the defining features of the Escherichia coli DsbA enzyme is its destabilizing disulfide, and this is also present in TcpG. The data presented here provide new insights into the structure and redox properties of this enzyme, showing that the binding mode identified between E. coli DsbB and DsbA is likely to be conserved in TcpG and that the β5-α7 loop near the proposed DsbB binding site is flexible, and suggesting that the tense oxidized conformation of TcpG may be the consequence of a short contact at the active site that is induced by disulfide formation and is relieved by reduction.

Interaction between Plate Make and Protein in Protein Crystallisation Screening

Background Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate. Methodology/Principal Findings We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. Conclusions/Significance Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallise, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.

Reciprocal allosteric regulation of p38γ and PTPN3 involves a PDZ domain-modulated complex formation.

Structural analysis of a phosphatase-kinase complex defines a role for the PDZ domain in regulating kinase inactivation. Structural Insights into Kinase and Phosphatase Regulation Unlike other members of the p38 family of kinases, p38γ has a PDZ-interacting motif. Phosphorylation of a tyrosine and a threonine in the kinase domain activates p38γ. The PDZ domain of the tyrosine phosphatase PTPN3 interacts with p38γ and is important for efficient dephosphorylation and inactivation of this kinase. Although Chen et al. discovered that the phosphatase domain of PTPN3 was sufficient to interact with dually phosphorylated p38γ, an interaction between the PDZ domain of PTPN3 and the PDZ-interacting motif of p38γ stabilized the complex and relieved an autoinhibitory intramolecular conformation of PTPN3. Structural analysis identified an invariant arginine in PTPN3 that interacted with the phosphorylated threonine in p38γ, suggesting that dephosphorylation of tyrosine by PTPN3 precedes dephosphorylation of threonine. These findings reveal new insight into the reciprocal allosteric regulation of PTPN3 and p38γ activity. The mitogen-activated protein kinase p38γ (also known as MAPK12) and its specific phosphatase PTPN3 (also known as PTPH1) cooperate to promote Ras-induced oncogenesis. We determined the architecture of the PTPN3-p38γ complex by a hybrid method combining x-ray crystallography, small-angle x-ray scattering, and chemical cross-linking coupled to mass spectrometry. A unique feature of the glutamic acid–containing loop (E-loop) of the phosphatase domain defined the substrate specificity of PTPN3 toward fully activated p38γ. The solution structure revealed the formation of an active-state complex between p38γ and the phosphatase domain of PTPN3. The PDZ domain of PTPN3 stabilized the active-state complex through an interaction with the PDZ-binding motif of p38γ. This interaction alleviated autoinhibition of PTPN3, enabling efficient tyrosine dephosphorylation of p38γ. Our findings may enable structure-based drug design targeting the PTPN3-p38γ interaction as an anticancer therapeutic.

The structure of the caspase recruitment domain of BinCARD reveals that all three cysteines can be oxidized

The caspase recruitment domain (CARD) is present in death-domain superfamily proteins involved in inflammation and apoptosis. BinCARD is named for its ability to interact with Bcl10 and inhibit downstream signalling. Human BinCARD is expressed as two isoforms that encode the same N-terminal CARD region but which differ considerably in their C-termini. Both isoforms are expressed in immune cells, although BinCARD-2 is much more highly expressed. Crystals of the CARD fold common to both had low symmetry (space group P1). Molecular replacement was unsuccessful in this low-symmetry space group and, as the construct contains no methionines, first one and then two residues were engineered to methionine for MAD phasing. The double-methionine variant was produced as a selenomethionine derivative, which was crystallized and the structure was solved using data measured at two wavelengths. The crystal structures of the native and selenomethionine double mutant were refined to high resolution (1.58 and 1.40 Å resolution, respectively), revealing the presence of a cis-peptide bond between Tyr39 and Pro40. Unexpectedly, the native crystal structure revealed that all three cysteines were oxidized. The mitochondrial localization of BinCARD-2 and the susceptibility of its CARD region to redox modification points to the intriguing possibility of a redox-regulatory role.

Science Signaling Podcast: 14 October 2014

The kinase p38γ and the phosphatase PTPN3 allosterically regulate one anothers activity. This Podcast features an interview with Kai-En Chen, Tzu-Ching Meng, and Andrew Wang, authors of a Research Article that appears in the 14 October 2014 issue of Science Signaling, about their crystal structure of the MAP kinase p38γ bound to the phosphatase that inactivates it. When activated, members of the Ras superfamily of small GTPases promote cell survival and stimulate cell proliferation, and mutations that cause overactivation of Ras can cause cells to become cancerous. Many mitogen-activated protein kinases (MAPKs) cooperate with Ras to promote oncogenesis. However, the phosphorylated, active form of the MAPK p38γ suppresses Ras-induced oncogenesis, whereas the unphosphorylated, inactive form of p38γ promotes Ras-induced oncogensis. Chen et al. solved the crystal structure of p38γ bound to the phosphatase that inactivates it, PTPN3.

Structural insights into the architecture and membrane interactions of the conserved COMMD proteins

The COMMD proteins are a conserved family of proteins with central roles in intracellular membrane trafficking and transcription. They form oligomeric complexes with each other and act as components of a larger assembly called the CCC complex, which is localized to endosomal compartments and mediates the transport of several transmembrane cargos. How these complexes are formed however is completely unknown. Here, we have systematically characterised the interactions between human COMMD proteins, and determined structures of COMMD proteins using X-ray crystallography and X-ray scattering to provide insights into the underlying mechanisms of homo- and heteromeric assembly. All COMMD proteins possess an α-helical N-terminal domain, and a highly conserved C-terminal domain that forms a tightly interlocked dimeric structure responsible for COMMD-COMMD interactions. The COMM domains also bind directly to components of CCC and mediate non-specific membrane association. Overall these studies show that COMMD proteins function as obligatory dimers with conserved domain architectures.