Structure (v.18, #5)
In This Issue (v-vi).
RZZ Finds Its Ancestral Roots by Alexandra Menant; Roger Karess (549-550).
In this issue of Structure, describe an unexpected structural similarity between the kinetochore component Rod-Zw10-Zwilch (RZZ) and the vesicle transport component Nag-Rint1-Zw10 (NRZ).
Class II Cytokine Common Receptors: Something Old, Something New by Andrew P. Hinck (551-552).
In this issue of Structure, Yoon and colleagues provide models of a low affinity cytokine common receptor, IL-10R2, in a ternary complex with two class II cytokines and their corresponding ligand-specific receptors, revealing the nature of their promiscuous interactions.
Local and Global Mobility in the ClpA AAA+ Chaperone Detected by Cryo-Electron Microscopy: Functional Connotations by Grégory Effantin; Takashi Ishikawa; Gian Marco De Donatis; Michael R. Maurizi; Alasdair C. Steven (553-562).
The ClpA chaperone combines with the ClpP peptidase to perform targeted proteolysis in the bacterial cytoplasm. ClpA monomer has an N-terminal substrate-binding domain and two AAA+ ATPase domains (D1 and D2). ClpA hexamers stack axially on ClpP heptamers to form the symmetry-mismatched protease. We used cryo-electron microscopy to visualize the ClpA-ATPγS hexamer, in the context of ClpAP complexes. Two segments lining the axial channel show anomalously low density, indicating that these motifs, which have been implicated in substrate translocation, are mobile. We infer that ATP hydrolysis is accompanied by substantial structural changes in the D2 but not the D1 tier. The entire N domain is rendered invisible by large-scale fluctuations. When deletions of 10 and 15 residues were introduced into the linker, N domain mobility was reduced but not eliminated and changes were observed in enzymatic activities. Based on these observations, we present a pseudo-atomic model of ClpAP holoenzyme, a dynamic proteolytic nanomachine.► The ClpAP unfoldase/peptidase is a dynamic proteolytic nanomachine ► N domain fluctuations are dampened but not eliminated by a 15 residue linker deletion ► The D2 “diaphragm loops” lining the axial channel are also mobile ► The D1 tier is a static platform on which the D2 domains shift upon ATP hydrolysis
Keywords: PROTEINS; CELLBIO; CELLCYCLE;
A Single Mutation Promotes Amyloidogenicity through a Highly Promiscuous Dimer Interface by Francis C. Peterson; Elizabeth M. Baden; Barbara A.L. Owen; Brian F. Volkman; Marina Ramirez-Alvarado (563-570).
Light chain amyloidosis is a devastating protein misfolding disease characterized by the accumulation of amyloid fibrils that causes tissue damage and organ failure. These fibrils are composed of monoclonal light chain protein secreted from an abnormal proliferation of bone marrow plasma cells. We previously reported that amyloidogenic light chain protein AL-09 adopts an altered dimer while its germline protein (κI O18/O8) forms a canonical dimer observed in other light chain crystal structures. In solution, conformational heterogeneity obscures all NMR signals at the AL-09 and κI O18/O8 dimer interfaces, so we solved the nuclear magnetic resonance structure of two related mutants. AL-09 H87Y adopts the normal dimer interface, but the κI Y87H solution structure presents an altered interface rotated 180° relative to the canonical dimer interface and 90° from the AL-09 arrangement. Our results suggest that promiscuity in the light chain dimer interface may promote new intermolecular contacts that may contribute to amyloid fibril structure.Display Omitted► Amyloidogenic light chains adopt altered dimer interface conformations ► Interface mutations destabilize canonical dimer arrangement ► Dynamic dimer interactions promote new contacts and amyloid formation ► Tyr-to-His substitution at position 87 promotes altered dimer and amyloidogenesis
Structure of an Apoptosome-Procaspase-9 CARD Complex by Shujun Yuan; Xinchao Yu; Maya Topf; Steven J. Ludtke; Xiaodong Wang; Christopher W. Akey (571-583).
Apaf-1 coassembles with cytochrome c to form the apoptosome, which then binds and activates procaspase-9 (pc-9). We removed pc-9 catalytic domains from the holoapoptosome by site-directed thrombinolysis. A structure of the resulting apoptosome-pc-9 CARD complex was then determined at ∼9.5 Å resolution. In our model, the central hub is constructed like other AAA+ protein rings but also contains novel features. At higher radius, the regulatory region of each Apaf-1 is comprised of tandem seven and eight blade β-propellers with cytochrome c docked between them. Remarkably, Apaf-1 CARDs are disordered in the ground state. During activation, each Apaf-1 CARD interacts with a pc-9 CARD and these heterodimers form a flexibly tethered “disk” that sits above the central hub. When taken together, the data reveal conformational changes during Apaf-1 assembly that allow pc-9 activation. The model also provides a plausible explanation for the effects of NOD mutations that have been mapped onto the central hub.► The human apoptosome is constructed like AAA+ ATPase rings but with novel features ► Apoptosome assembly releases flexibly linked, N-terminal CARDs that bind pc-9 ► Apaf-1 CARDs and pc-9 CARDs form a disk above the apoptosome that may activate pc-9 ► A subnanometer model provides insights into assembly and function of NOD proteins
Keywords: PROTEINS; SIGNALLING; CELLBIO;
The Prp19 WD40 Domain Contains a Conserved Protein Interaction Region Essential for Its Function by Craig W. Vander Kooi; Liping Ren; Ping Xu; Melanie D. Ohi; Kathleen L. Gould; Walter J. Chazin (584-593).
Prp19 is a member of the WD40 repeat family of E3 ubiquitin ligases and a conserved eukaryotic RNA splicing factor essential for activation and stabilization of the spliceosome. To understand the role of the WD40 repeat domain of Prp19 we have determined its structure using X-ray crystallography. The domain has a distorted seven bladed WD40 architecture with significant asymmetry due to irregular packing of blades one and seven into the core of the WD40 domain. Structure-based mutagenesis identified a highly conserved surface centered around blade five that is required for the physical interaction between Prp19 and Cwc2, another essential splicing factor. This region is found to be required for Prp19 function and yeast viability. Experiments in vitro and in vivo demonstrate that two molecules of Cwc2 bind to the Prp19 tetramer. These coupled structural and functional studies provide a model for the functional architecture of Prp19.► The Prp19 WD40 domain adopts a distorted seven bladed β propeller fold ► A conserved surface patch is required for interaction with Cwc2 and for in vivo function ► Prp19 forms a 4:2 complex with Cwc2 in vitro and in vivo
Keywords: PROTEINS; CELLBIO; RNA;
Structure of Arabidopsis HYPONASTIC LEAVES1 and Its Molecular Implications for miRNA Processing by Seong Wook Yang; Hong-Ying Chen; Jing Yang; Satoru Machida; Nam-Hai Chua; Y. Adam Yuan (594-605).
The Arabidopsis HYPONASTIC LEAVES1 (HYL1) is a double-stranded RNA-binding protein that forms a complex with DICER-LIKE1 (DCL1) and SERRATE to facilitate processing of primary miRNAs into microRNAs (miRNAs). However, the structural mechanisms of miRNA maturation by this complex are poorly understood. Here, we present the crystal structures of double-stranded RNA binding domains (dsRBD1 and dsRBD2) of HYL1 and HYL1 dsRBD1 (HR1)/dsRNA complex as well as human TRBP2 dsRBD2 (TR2)/dsRNA complex for comparison analysis. Structural and functional study demonstrates that both HR1 and TR2 are canonical dsRBDs for dsRNA binding, whereas HR2 of HYL1 is a non-canonical dsRBD harboring a putative dimerization interface. Domain swapping within the context of HYL1 demonstrates that TR2 can supplant the function of HR1 in vitro and in vivo. Further biochemical analyses suggest that HYL1 probably binds to the miRNA/miRNA∗ region of precursors as a dimer mediated by HR2.► Crystal structures of dsRBD1 and dsRBD2 of HYL1 (HR1 and HR2) and dsRBD2 of TRBP2 (TR2) ► Crystal structures of HR1/dsRNA and TR2/dsRNA complexes ► Chimerical TR2-HR2 protein rescues HYL1 function in vitro and in vivo ► HYL1 probably binds to miRNA/miRNA∗ region of precursors as a dimer mediated by HR2
The Structure of PknB Extracellular PASTA Domain from Mycobacterium tuberculosis Suggests a Ligand-Dependent Kinase Activation by Philippe Barthe; Galina V. Mukamolova; Christian Roumestand; Martin Cohen-Gonsaud (606-615).
PknB is a transmembrane Ser/Thr protein kinase that defines and belongs to an ultraconserved kinase subfamily found in Gram-positive bacteria. Essential for Mycobacterium tuberculosis growth, its close homolog in Bacillus subtilis has been linked to exit from dormancy. The kinase possesses an extracellular region composed of a repetition of PASTA domains, believed to bind peptidoglycan fragments that might act as a signaling molecule. We report here the first solution structure of this extracellular region. Small-angle X-ray scattering and nuclear magnetic resonance studies show that the four PASTA domains display an unexpected linear organization, contrary to what is observed in the distant protein PBP2x from Streptococccus pneumoniae where two PASTA domains fold over in a compact structure. We propose a model for PknB activation based on a ligand-dependent dimerization of the extracellular PASTA domains that initiates multiple signaling pathways.► The Ser/Thr protein kinase PknB posses an external domain composed of four PASTA modules ► PknB Bacillus subtilis homolog is a receptor that promotes the exit of dormancy ► First structure of the kinase external PASTA domain ► Unexpected linear organization for the modules suggest ligand-dependent dimerization
Keywords: PROTEINS; MICROBIO;
Structural Analysis of the RZZ Complex Reveals Common Ancestry with Multisubunit Vesicle Tethering Machinery by Filiz Çivril; Annemarie Wehenkel; Federico M. Giorgi; Stefano Santaguida; Andrea Di Fonzo; Gabriela Grigorean; Francesca D. Ciccarelli; Andrea Musacchio (616-626).
The RZZ complex recruits dynein to kinetochores. We investigated structure, topology, and interactions of the RZZ subunits (ROD, ZWILCH, and ZW10) in vitro, in vivo, and in silico. We identify neuroblastoma-amplified gene (NAG), a ZW10 binder, as a ROD homolog. ROD and NAG contain an N-terminal β propeller followed by an α solenoid, which is the architecture of certain nucleoporins and vesicle coat subunits, suggesting a distant evolutionary relationship. ZW10 binding to ROD and NAG is mutually exclusive. The resulting ZW10 complexes (RZZ and NRZ) respectively contain ZWILCH and RINT1 as additional subunits. The X-ray structure of ZWILCH, the first for an RZZ subunit, reveals a novel fold distinct from RINT1's. The evolutionarily conserved NRZ likely acts as a tethering complex for retrograde trafficking of COPI vesicles from the Golgi to the endoplasmic reticulum. The RZZ, limited to metazoans, probably evolved from the NRZ, exploiting the dynein-binding capacity of ZW10 to direct dynein to kinetochores.► The ZW10 protein is shown to be part of two structurally related complexes, the RZZ and the NRZ ► ROD and NAG, subunits of the RZZ and NRZ, respectively, consist of a β propeller and an α solenoid ► This organization is typical of clathrin, COP-I, and nucleoporins, suggesting the RZZ evolved from them ► A crystal structure of ZWILCH, an RZZ subunit, is reported and shown to have a novel fold
Keywords: PROTEINS; CELLBIO;
Recognition of the Regulatory Nascent Chain TnaC by the Ribosome by Leonardo G. Trabuco; Christopher B. Harrison; Eduard Schreiner; Klaus Schulten (627-637).
Regulatory nascent chains interact with the ribosomal exit tunnel and modulate their own translation. To characterize nascent chain recognition by the ribosome at the atomic level, extensive molecular dynamics simulations of TnaC, the leader peptide of the tryptophanase operon, inside the exit tunnel were performed for an aggregate time of 2.1 μs. The simulations, complemented by quantum chemistry calculations, suggest that the critical TnaC residue W12 is recognized by the ribosome via a cation-π interaction, whereas TnaC's D16 forms salt bridges with ribosomal proteins. The simulations also show that TnaC-mediated translational arrest does not involve a swinging of ribosomal protein L22, as previously proposed. Furthermore, bioinformatic analyses and simulations suggest nascent chain elements that may prevent translational arrest in various organisms. Altogether, the current study unveils atomic-detail interactions that explain the role of elements of TnaC and the ribosome essential for translational arrest.Display Omitted► Residue W12 of TnaC is recognized by the ribosome via a cation-π interaction ► Residue D16 of TnaC forms salt bridges with ribosomal proteins ► TnaC-mediated stalling does not involve a swinging of ribosomal protein L22 ► TnaC mutations I19R and V20R are predicted to reduce translational arrest
Structure and Mechanism of Receptor Sharing by the IL-10R2 Common Chain by Sung-il Yoon; Brandi C. Jones; Naomi J. Logsdon; Bethany D. Harris; Ashlesha Deshpande; Svetlana Radaeva; Brian A. Halloran; Bin Gao; Mark R. Walter (638-648).
IL-10R2 is a shared cell surface receptor required for the activation of five class 2 cytokines (IL-10, IL-22, IL-26, IL-28, and IL-29) that play critical roles in host defense. To define the molecular mechanisms that regulate its promiscuous binding, we have determined the crystal structure of the IL-10R2 ectodomain at 2.14 Å resolution. IL-10R2 residues required for binding were identified by alanine scanning and used to derive computational models of IL-10/IL-10R1/IL-10R2 and IL-22/IL-22R1/IL-10R2 ternary complexes. The models reveal a conserved binding epitope that is surrounded by two clefts that accommodate the structural and chemical diversity of the cytokines. These results provide a structural framework for interpreting IL-10R2 single nucleotide polymorphisms associated with human disease.► The crystal structure of the IL-10R2 is distinct from IL-10R1 and IL-22R1 chains ► IL-10R2 residues required for binding multiple cytokines have been identified ► Docking studies provide structures of the promiscuous recognition paradigm ► Class 1 and class 2 receptor common chains exhibit conserved binding epitopes
Two-State Conformations in the Hyaluronan-Binding Domain Regulate CD44 Adhesiveness under Flow Condition by Shinji Ogino; Noritaka Nishida; Ryo Umemoto; Miho Suzuki; Mitsuhiro Takeda; Hiroaki Terasawa; Joji Kitayama; Masanori Matsumoto; Haruko Hayasaka; Masayuki Miyasaka; Ichio Shimada (649-656).
The hyaluronan (HA) receptor CD44 mediates cell adhesion in leukocyte trafficking and tumor metastasis. Our previous nuclear magnetic resonance (NMR) studies revealed that the CD44 hyaluronan-binding domain (HABD) alters its conformation upon HA binding, from the ordered (O) to the partially disordered (PD) conformation. Here, we demonstrate that the HABD undergoes an equilibrium between the O and PD conformations, in either the presence or absence of HA, which explains the seemingly contradictory X-ray and NMR structures of the HA-bound HABD. An HABD mutant that exclusively adopts the PD conformation displayed a higher HA affinity than the wild-type. Rolling of the cells expressing the mutant CD44 was less efficient than those expressing the wild-type, due to the decreased tether frequency and the slow cellular off rate. Considering that the mutant CD44, devoid of the low-affinity state, exhibited impaired rolling, we conclude that the coexistence of the high- and low-affinity states of the HABD is essential for the CD44-mediated rolling.Display Omitted► CD44 HABD exists in two-state equilibrium either in the ligand-free or bound states ► Ligand-binding shifts the equilibrium from the O form to the PD form ► O and PD form represents low- and high-affinity states for ligand, respectively ► Two-state equilibrium is crucial for CD44-mediated cell rolling under flow condition
Keywords: PROTEINS; CELLBIO; MOLIMMUNO;