Crystal Structure of the GerBC Component of a Bacillus subtilis Spore Germinant Receptor

The nutrient germinant receptors (nGRs) of spores of Bacillus species are clusters of three proteins that play a critical role in triggering the germination of dormant spores in response to specific nutrient molecules. Here, we report the crystal structure of the C protein of the GerB germinant receptor, so-called GerBC, of Bacillus subtilis spores at 2.3 Å resolution. The GerBC protein adopts a previously uncharacterized type of protein fold consisting of three distinct domains, each of which is centered by a β sheet surrounded by multiple α helices. Secondary-structure prediction and structure-based sequence alignment suggest that the GerBC structure represents the prototype for C subunits of nGRs from spores of all Bacillales and Clostridiales species and defines two highly conserved structural regions in this family of proteins. GerBC forms an interlocked dimer in the crystalline state but is predominantly monomeric in solution, pointing to the possibility that GerBC oligomerizes as a result of either high local protein concentrations or interaction with other nGR proteins in spores. Our findings provide the first structural view of the nGR subunits and a molecular framework for understanding the architecture, conservation, and function of nGRs.     

High-resolution crystal structure reveals a HEPN domain at the C-terminal region of S. cerevisiae RNA endonuclease Swt1

Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomyces cerevisiae, which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance.     

Structure of the Entire Stalk Region of the Dynein Motor Domain

Dyneins are large microtubule-based motor complexes that power a range of cellular processes including the transport of organelles, as well as the beating of cilia and flagella. The motor domain is located within the dynein heavy chain and comprises an N-terminal mechanical linker element, a central ring of six AAA + modules of which four bind or hydrolyze ATP, and a long stalk extending from the AAA + ring with a microtubule-binding domain (MTBD) at its tip. A crucial mechanism underlying the motile activity of cytoskeletal motor proteins is precise coupling between the ATPase and track-binding activities. In dynein, a stalk region consisting of a long (~ 15 nm) antiparallel coiled coil separates these two activities, which must facilitate communication between them. This communication is mediated by a small degree of helix sliding in the coiled coil. However, no high-resolution structure is available of the entire stalk region including the MTBD. Here, we have reported the structure of the entire stalk region of mouse cytoplasmic dynein in a weak microtubule-binding state, which was determined using X-ray crystallography, and have compared it with the dynein motor domain from Dictyostelium discoideum in a strong microtubule-binding state and with a mouse MTBD with its distal portion of the coiled coil fused to seryl-tRNA synthetase from Thermus thermophilus. Our results strongly support the helix-sliding model based on the complete structure of the dynein stalk with a different form of coiled-coil packing. We also propose a plausible mechanism of helix sliding together with further analysis using molecular dynamics simulations. Our results present the importance of conserved proline residues for an elastic motion of stalk coiled coil and imply the manner of change between high-affinity state and low-affinity state of MTBD.     

Crystal Structure of the C-Terminal Domain of Human DPY-30-Like Protein: A Component of the Histone Methyltransferase Complex

The conserved DPY-30 is an essential component of the dosage compensation complex that balances the X-linked gene expression by regulation of the complex formation in Caenorhabditis elegans. The human DPY-30-like protein (DPY-30L) homolog is a conserved member of certain histone methyltransferase (HMT) complexes. In the human MLL1 (mixed-lineage leukemia-1) HMT complex, DPY-30L binds to the BRE2 homolog ASH2L in order to regulate histone 3-lysine 4 trimethylation. We have determined the 1.2-Å crystal structure of the human DPY-30L C-terminal domain (DPY-30L_C). The DPY-30L_C structure, harboring the conserved DPY-30 motif, is composed of two α-helices linked by a sharp loop and forms a typical X-type four-helix bundle required for dimer formation. DPY-30L_C dimer formation is largely mediated by an extensive hydrophobic interface with some additional polar interactions. The oligomerization of DPY-30L_C in solution, together with its reported binding to ASH2L, leads us to propose that the hydrophobic surface of the dimer may provide a platform for interaction with ASH2L in the MLL1 HMT complex.     

Crystal Structure of a Periplasmic Substrate-Binding Protein in Complex with Calcium Lactate

Lactate is utilized in many biological processes, and its transport across biological membranes is mediated with various types of transporters. Here, we report the crystal structures of a lactate-binding protein of a TRAP (tripartite ATP-independent periplasmic) secondary transporter from Thermus thermophilus HB8. The folding of the protein is typical for a type II periplasmic solute-binding protein and forms a dimer in a back-to-back manner. One molecule of l-lactate is clearly identified in a cleft of the protein as a complex with a calcium ion. Detailed crystallographic and biochemical analyses revealed that the calcium ion can be removed from the protein and replaced with other divalent cations. This characterization of the structure of a protein binding with calcium lactate makes a significant contribution to our understanding of the mechanisms by which calcium and lactate are accommodated in cells.     

Crystal Structure of the Electron Carrier Domain of the Reaction Center Cytochrome cz Subunit from Green Photosynthetic Bacterium Chlorobium tepidum

In green sulfur photosynthetic bacteria, the cytochrome c_z (cyt c_z) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt c_z subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt c_z subunit (C-cyt c_z) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-Å resolution. The overall fold of C-cyt c_z consists of four α-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt c_z supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt c_z.     

Model for Substrate Interactions in C5a Peptidase from Streptococcus pyogenes: A 1.9 Å Crystal Structure of the Active Form of ScpA

The crystal structure of an active form of ScpA has been solved to 1.9 Å resolution. ScpA is a multidomain cell-envelope subtilase from Streptococcus pyogenes that cleaves complement component C5a. The catalytic triad of ScpA is geometrically consistent with other subtilases, clearly demonstrating that the additional activation mechanism proposed for the Streptococcus agalactiae homologue (ScpB) is not required for ScpA. The ScpA structure revealed that access to the catalytic site is restricted by variable regions in the catalytic domain (vr7, vr9, and vr11) and by the presence of the inserted protease-associated (PA) domain and the second fibronectin type III domains (Fn2). Modeling of the ScpA-C5a complex indicates that the substrate binds with carboxyl-terminal residues (65-74) extended through the active site and core residues (1-64) forming exosite-type interactions with the Fn2 domain. This is reminiscent of the two-site mechanism proposed for C5a binding to its receptor. In the nonprime region of the active site, interactions with the substrate backbone are predicted to be more similar to those observed in kexins, involving a single β-strand in the peptidase. However, in contrast to kexins, there would be diminished emphasis on side-chain interactions, with little charged character in the S3-S1 and S6-S4 subsites occupied by the side chains of residues in vr7 and vr9. Substrate binding is anticipated to be dominated by ionic interactions in two distinct regions of ScpA. On the prime side of the active site, salt bridges are predicted between P1′, P2′, and P7′ residues, and residues in the catalytic and PA domains. Remote to the active site, a larger number of ionic interactions between residues in the C5a core and the Fn2 domain are observed in the model. Thus, both PA and Fn2 domains are expected to play significant roles in substrate recognition.     

Crystal Structure of the Membrane Fusion Protein CusB from Escherichia coli

Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein (MFP) that is critical for substrate transport. We here present the x-ray structures of the CusB MFP from the copper/silver efflux system of E. coli. This is the first structure of any MFPs associated with heavy-metal efflux transporters. CusB bridges the inner-membrane efflux pump CusA and outer-membrane channel CusC to mediate resistance to Cu⁺ and Ag⁺ ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly β-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of MFPs, the α-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N- and C-termini of CusB form the first β-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu⁺ and Ag⁺ were revealed by the crystals of the CusB-Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of an MFP in the resistance-nodulation-division efflux system and provide direct evidence that this protein specifically interacts with transported substrates.     

Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-Å X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.     

Crystal structure of a rigid four-spectrin-repeat fragment of the human desmoplakin plakin domain

The plakin protein family serves to connect cell-cell and cell-matrix adhesion molecules to the intermediate filament cytoskeleton. Desmoplakin (DP) is an integral part of desmosomes, where it links desmosomal cadherins to the intermediate filaments. The 1056-amino-acid N-terminal region of DP contains a plakin domain common to members of the plakin family. Plakin domains contain multiple copies of spectrin repeats (SRs). We determined the crystal structure of a fragment of DP, residues 175-630, consisting of four SRs and an inserted SH3 domain. The four repeats form an elongated, rigid structure. The SH3 domain is present in a loop between two helices of an SR and interacts extensively with the preceding SR in a manner that appears to limit inter-repeat flexibility. The intimate intramolecular association of the SH3 domain with the preceding SR is also observed in plectin, another plakin protein, but not in α-spectrin, suggesting that the SH3 domain of plakins contributes to the stability and rigidity of this subfamily of SR-containing proteins.     

Two Complementary Enzymes for Threonylation of tRNA in Crenarchaeota: Crystal Structure of Aeropyrum pernix Threonyl-tRNA Synthetase Lacking a cis-Editing Domain

In protein synthesis, threonyl-tRNA synthetase (ThrRS) must recognize threonine (Thr) from the 20 kinds of amino acids and the cognate tRNA^Thr from different tRNAs in order to generate Thr-tRNA^Thr. In general, an organism possesses one kind of gene corresponding to ThrRS. However, it has been recently found that some organisms have two different genes for ThrRS in the genome, suggesting that their proteins ThrRS-1 and ThrRS-2 function separately and complement each other in the threonylation of tRNA^Thr, one for catalysis and the other for trans-editing of misacylated Ser-tRNA^Thr. In order to clarify their three-dimensional structures, we performed X-ray analyses of two putatively assigned ThrRSs from Aeropyrum pernix (ApThrRS-1 and ApThrRS-2). These proteins were overexpressed in Escherichia coli, purified, and crystallized. The crystal structure of ApThrRS-1 has been successfully determined at 2.3 Å resolution. ApThrRS-1 is a dimeric enzyme composed of two identical subunits, each containing two domains for the catalytic reaction and for anticodon binding. The essential editing domain is completely missing as expected. These structural features reveal that ThrRS-1 catalyzes only the aminoacylation of the cognate tRNA, suggesting the necessity of the second enzyme ThrRS-2 for trans-editing. Since the N-terminal sequence of ApThrRS-2 is similar to the sequence of the editing domain of ThrRS from Pyrococcus abyssi, ApThrRS-2 has been expected to catalyze deaminoacylation of a misacylated serine moiety at the CCA terminus.     

An Antibody against the C-Terminal Domain of PCSK9 Lowers LDL Cholesterol Levels In Vivo

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with autosomal dominant hypercholesterolemia, a state of elevated levels of LDL (low-density lipoprotein) cholesterol. Autosomal dominant hypercholesterolemia can result in severe implications such as stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the epidermal growth factor-like repeat A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Although the antibody does not inhibit binding of PCSK9 to epidermal growth factor-like repeat A, it partially reverses PCSK9-induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.     

Structure of a polyisoprenoid binding domain from Saccharophagus degradans implicated in plant cell wall breakdown

Saccharophagus degradans belongs to a recently discovered group of marine bacteria equipped with an arsenal of sugar cleaving enzymes coupled to carbohydrate-binding domains to degrade various insoluble complex polysaccharides. The modular Sde-1182 protein consists of a family 2 carbohydrate binding module linked to a X158 domain of unknown function. The 1.9 Å and 1.55 Å resolution crystal structures of the isolated X158 domain bound to the two related polyisoprenoid molecules, ubiquinone and octaprenyl pyrophosphate, unveil a β-barrel architecture reminiscent of the YceI-like superfamily that resembles the architecture of the lipocalin fold. This unprecedented association coupling oxidoreduction and carbohydrate recognition events may have implications for effective nutrient uptake in the marine environment.