Lichuan Gu

Structure of the CED-4-CED-9 complex provides insights into programmed cell death in Caenorhabditis elegans

Interplay among four genes—egl-1, ced-9, ced-4 and ced-3—controls the onset of programmed cell death in the nematode Caenorhabditis elegans. Activation of the cell-killing protease CED-3 requires CED-4. However, CED-4 is constitutively inhibited by CED-9 until its release by EGL-1. Here we report the crystal structure of the CED-4–CED-9 complex at 2.6u2009Å resolution, and a complete reconstitution of the CED-3 activation pathway using homogeneous proteins of CED-4, CED-9 and EGL-1. One molecule of CED-9 binds to an asymmetric dimer of CED-4, but specifically recognizes only one of the two CED-4 molecules. This specific interaction prevents CED-4 from activating CED-3. EGL-1 binding induces pronounced conformational changes in CED-9 that result in the dissociation of the CED-4 dimer from CED-9. The released CED-4 dimer further dimerizes to form a tetramer, which facilitates the autoactivation of CED-3. Together, our studies provide important insights into the regulation of cell death activation in C. elegans.

Structural analysis of a rhomboid family intramembrane protease reveals a gating mechanism for substrate entry.

Intramembrane proteolysis regulates diverse biological processes. Cleavage of substrate peptide bonds within the membrane bilayer is catalyzed by integral membrane proteases. Here we report the crystal structure of the transmembrane core domain of GlpG, a rhomboid-family intramembrane serine protease from Escherichia coli. The protein contains six transmembrane helices, with the catalytic Ser201 located at the N terminus of helix α4 approximately 10 Å below the membrane surface. Access to water molecules is provided by a central cavity that opens to the extracellular region and converges on Ser201. One of the two GlpG molecules in the asymmetric unit has an open conformation at the active site, with the transmembrane helix α5 bent away from the rest of the molecule. Structural analysis suggests that substrate entry to the active site is probably gated by the movement of helix α5.

Structure of the BRCT Repeats of BRCA1 Bound to a BACH1 Phosphopeptide: Implications for Signaling

The recognition of the phosphorylated BACH1 helicase by the BRCA1 C-terminal (BRCT) repeats is important to the tumor suppressor function of BRCA1. Here we report the crystal structure of the BRCT repeats of human BRCA1 bound to a phosphorylated BACH1 peptide at 2.3 A resolution. The phosphorylated serine 990 and phenylalanine 993 of BACH1 anchor the binding to BRCA1 through specific interactions with a surface cleft at the junction of the two BRCT repeats. This surface cleft is highly conserved in BRCA1 across species, suggesting an evolutionarily conserved function of phosphopeptide recognition. Importantly, conserved amino acids critical for BACH1 binding are frequently targeted for missense mutations in breast cancer. These mutations greatly diminish the ability of BRCA1 to interact with the phosphorylated BACH1 peptide. Additional structural analysis revealed significant implications for understanding the function of the BRCT family of proteins in DNA damage and repair signaling.

Structural Basis of Competitive Recognition of p53 and MDM2 by HAUSP/USP7: Implications for the Regulation of the p53-MDM2 Pathway.

Herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7), a deubiquitylating enzyme of the ubiquitin-specific processing protease family, specifically deubiquitylates both p53 and MDM2, hence playing an important yet enigmatic role in the p53–MDM2 pathway. Here we demonstrate that both p53 and MDM2 specifically recognize the N-terminal tumor necrosis factor–receptor associated factor (TRAF)–like domain of HAUSP in a mutually exclusive manner. HAUSP preferentially forms a stable HAUSP–MDM2 complex even in the presence of excess p53. The HAUSP-binding elements were mapped to a peptide fragment in the carboxy-terminus of p53 and to a short-peptide region preceding the acidic domain of MDM2. The crystal structures of the HAUSP TRAF-like domain in complex with p53 and MDM2 peptides, determined at 2.3-Å and 1.7-Å resolutions, respectively, reveal that the MDM2 peptide recognizes the same surface groove in HAUSP as that recognized by p53 but mediates more extensive interactions. Structural comparison led to the identification of a consensus peptide-recognition sequence by HAUSP. These results, together with the structure of a combined substrate-binding-and-deubiquitylation domain of HAUSP, provide important insights into regulation of the p53–MDM2 pathway by HAUSP.

Molecular Mechanism of AHSP-Mediated Stabilization of α-Hemoglobin

Hemoglobin A (HbA), the oxygen delivery system in humans, comprises two alpha and two beta subunits. Free alpha-hemoglobin (alphaHb) is unstable, and its precipitation contributes to the pathophysiology of beta thalassemia. In erythrocytes, the alpha-hemoglobin stabilizing protein (AHSP) binds alphaHb and inhibits its precipitation. The crystal structure of AHSP bound to Fe(II)-alphaHb reveals that AHSP specifically recognizes the G and H helices of alphaHb through a hydrophobic interface that largely recapitulates the alpha1-beta1 interface of hemoglobin. The AHSP-alphaHb interactions are extensive but suboptimal, explaining why beta-hemoglobin can competitively displace AHSP to form HbA. Remarkably, the Fe(II)-heme group in AHSP bound alphaHb is coordinated by the distal but not the proximal histidine. Importantly, binding to AHSP facilitates the conversion of oxy-alphaHb to a deoxygenated, oxidized [Fe(III)], nonreactive form in which all six coordinate positions are occupied. These observations reveal the molecular mechanisms by which AHSP stabilizes free alphaHb.

C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1

Externalization of phosphatidylserine, which is normally restricted to the inner leaflet of plasma membrane, is a hallmark of mammalian apoptosis. It is not known what activates and mediates the phosphatidylserine externalization process in apoptotic cells. Here, we report the development of an annexin V-based phosphatidylserine labelling method and show that a majority of apoptotic germ cells in Caenorhabditis elegans have surface-exposed phosphatidylserine, indicating that phosphatidylserine externalization is a conserved apoptotic event in worms. Importantly, inactivation of the gene encoding either the C. elegans apoptosis-inducing factor (AIF) homologue (WAH-1), a mitochondrial apoptogenic factor, or the C. elegans phospholipid scramblase 1 (SCRM-1), a plasma membrane protein, reduces phosphatidylserine exposure on the surface of apoptotic germ cells and compromises cell-corpse engulfment. WAH-1 associates with SCRM-1 and activates its phospholipid scrambling activity in vitro. Thus WAH-1, after its release from mitochondria during apoptosis, promotes plasma membrane phosphatidylserine externalization through its downstream effector, SCRM-1.

Structure of oxidized alpha-haemoglobin bound to AHSP reveals a protective mechanism for haem.

The synthesis of haemoglobin A (HbA) is exquisitely coordinated during erythrocyte development to prevent damaging effects from individual α- and β-subunits. The α-haemoglobin-stabilizing protein (AHSP) binds α-haemoglobin (αHb), inhibits the ability of αHb to generate reactive oxygen species and prevents its precipitation on exposure to oxidant stress. The structure of AHSP bound to ferrous αHb is thought to represent a transitional complex through which αHb is converted to a non-reactive, hexacoordinate ferric form. Here we report the crystal structure of this ferric αHb–AHSP complex at 2.4u2009Å resolution. Our findings reveal a striking bis-histidyl configuration in which both the proximal and the distal histidines coordinate the haem iron atom. To attain this unusual conformation, segments of αHb undergo drastic structural rearrangements, including the repositioning of several α-helices. Moreover, conversion to the ferric bis-histidine configuration strongly and specifically inhibits redox chemistry catalysis and haem loss from αHb. The observed structural changes, which impair the chemical reactivity of haem iron, explain how AHSP stabilizes αHb and prevents its damaging effects in cells.

Structural basis for activation and inhibition of the secreted chlamydia protease CPAF

The obligate intracellular pathogen Chlamydia trachomatis is the most common cause of sexually transmitted bacterial disease. It secretes a protease known as chlamydial protease/proteasome-like activity factor (CPAF) that degrades many host molecules and plays a major role in Chlamydia pathogenesis. Here, we show that mature CPAF is a homodimer of the catalytic domains, each of which comprises two distinct subunits. Dormancy of the CPAF zymogen is maintained by an internal inhibitory segment that binds the CPAF active site and blocks its homodimerization. CPAF activation is initiated by trans-autocatalytic cleavage, which induces homodimerization and conformational changes that assemble the catalytic triad. This assembly leads to two autocatalytic cleavages and removal of the inhibitory segment, enabling full CPAF activity. CPAF is covalently bound and inhibited by the proteasome inhibitor lactacystin. These results reveal the activation mechanism of the CPAF serine protease and suggest new opportunities for anti-Chlamydia drug development.

Structural basis for the catalytic mechanism of phosphothreonine lyase

Salmonella SpvC belongs to a new enzyme family designated phosphothreonine lyases that irreversibly inactivate mitogen-activated protein kinases. The crystal structure of SpvC reported here reveals that the two phosphorylated residues in the substrate peptide predominantly mediate its recognition by SpvC. Substrate-induced conformational changes in SpvC sequester the phosphothreonine in a completely solvent-free environment, preventing the hydrolysis of the phosphate group and facilitating the elimination reaction.

Structural Insights into KChIP4a Modulation of Kv4.3 Inactivation

Dynamic inactivation in Kv4 A-type K+ current plays a critical role in regulating neuronal excitability by shaping action potential waveform and duration. Multifunctional auxiliary KChIP1–4 subunits, which share a high homology in their C-terminal core regions, exhibit distinctive modulation of inactivation and surface expression of pore-forming Kv4 subunits. However, the structural differences that underlie the functional diversity of Kv channel-interacting proteins (KChIPs) remain undetermined. Here we have described the crystal structure of KChIP4a at 3.0Å resolution, which shows distinct N-terminal α-helices that differentiate it from other KChIPs. Biochemical experiments showed that competitive binding of the Kv4.3 N-terminal peptide to the hydrophobic groove of the core of KChIP4a causes the release of the KChIP4a N terminus that suppresses the inactivation of Kv4.3 channels. Electrophysiology experiments confirmed that the first N-terminal α-helix peptide (residues 1–34) of KChIP4a, either by itself or fused to N-terminal truncated Kv4.3, can confer slow inactivation. We propose that N-terminal binding of Kv4.3 to the core of KChIP4a mobilizes the KChIP4a N terminus, which serves as the slow inactivation gate.