Structural Homology between the C-Terminal Domain of the PapC Usher and Its Plug

P pili are extracellular appendages responsible for the targeting of uropathogenic Escherichia coli to the kidney. They are assembled by the chaperone-usher (CU) pathway of pilus biogenesis involving two proteins, the periplasmic chaperone PapD and the outer membrane assembly platform, PapC. Many aspects of the structural biology of the Pap CU pathway have been elucidated, except for the C-terminal domain of the PapC usher, the structure of which is unknown. In this report, we identify a stable and folded fragment of the C-terminal region of the PapC usher and determine its structure using both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. These structures reveal a β-sandwich fold very similar to that of the plug domain, a domain of PapC obstructing its translocation domain. This structural similarity suggests similar functions in usher-mediated pilus biogenesis, playing out at different stages of the process. This structure paves the way for further functional analysis targeting surfaces common to both the plug and the C-terminal domain of PapC.Adhesive surface organelles termed pili mediate the adhesion of bacteria to host cells. Pili assembled by the chaperone-usher (CU) pathway form one of five major classes of nonflagellar surface appendages in Gram-negative bacteria, with the P pilus system from uropathogenic Escherichia coli being one of the two best-characterized CU systems. These pili are multisubunit structures consisting of two distinct subassemblies, a rigid rod with a diameter of 6.8 nm and a distal flexible tip fibrillum with a diameter of 2 nm (18, 21). In P pili the helical rod is comprised of more than 1,000 copies of the PapA subunits arranged in a right-handed helical cylinder with 3.3 subunits per turn (3, 8, 14), and the tip fibrillum is comprised of 5 to 10 copies of the PapE subunits (21). Two “adaptor” subunits, PapK and PapF, connect the PapE tip fibrillum to the PapA rod and the PapE tip fibrillum to the distal PapG adhesin (16, 21). The proximal end of the pilus is terminated by the PapH subunit (2, 50). The PapG adhesin mediates the bacterial colonization of the kidney (25, 40) by binding to the globoseries of glycolipids present in the human kidney (25, 40) (Fig. ​(Fig.1A),1A), an event that is critical in pyelonephritis.Open in a separate windowFIG. 1.(A) Schematic diagram of a P pilus assembled in the usher translocation platform. Subunits are represented by oval shapes, and N-terminal extensions are represented by short rectangular shapes. The usher homodimer is represented in the outer membrane (OM). In the usher protomer through which the nascent pilus passes, two positions of the plug are indicated by P where the plug is positioned to the side of the transmembrane barrel''s lumen and P′ where the plug has swung into the periplasmic space. (B) Domain organization of the PapC usher based on amino acid sequence. The C-terminal domain sequences are indicated in marine blue. The constructs used in this study are schematically represented underneath; all converge to a fragment containing residues 722 to 809, termed the “PapC CTD.” Ntd, N-terminal domain. (C) Identification of a discrete folding unit at the C terminus of PapC. Shown is an SDS-PAGE gel stained with Coomassie blue of the eluted PapC C-terminal fragments obtained with a construct comprising residues 641 to 809 after the first purification step. PS, prestained protein standards; Inj, loaded sample; FT, flowthrough.The assembly of pili is a coordinated process requiring two proteins: a chaperone and an outer membrane assembly platform, the usher. Pilus subunits are translocated into the periplasm via the general secretory machinery (38, 47). The binding of the PapD chaperone to the nascently translocated subunits facilitates their folding on the chaperone template. The chaperone remains bound to the folded subunits capping their interactive surfaces, thus preventing nonproductive interactions in the periplasm (7). Chaperone-subunit complexes are then targeted to the usher (PapC), where subunits polymerize in an ordered fashion and translocate across the outer membrane through the usher pore (47, 52). Subunit folding and stabilization occur when the chaperone and subunit form a complex through a mechanism termed donor strand complementation (DSC) (9, 41). In this mechanism the C-terminally truncated Ig-like fold of the pilus subunits, which contains only six of the seven β-strands that constitute the canonical Ig fold, is complemented by the donation of a β-strand from the chaperone (9, 41). Chaperone-subunit complexes are then targeted to the outer membrane usher, where the chaperone is released and subunits are noncovalently joined to preceding subunits in the nascent pilus fiber. This polymerization process is made possible by the presence of a disordered N-terminal extension sequence (NTES) in each subunit (except the adhesin) (41), which during pilus assembly displaces the strand donated by the chaperone, thereby substituting for the missing secondary structure in the previously assembled subunit. This mechanism is called donor-strand exchange (DSE) (9, 41, 42, 55). It is believed that this structural reorganization provides the driving force for pilus biogenesis, since no ATP hydrolysis or other type of external energy source is required (17, 56).DSE occurs at the outer membrane usher, which acts as a catalyst for polymerization (34). Biophysical and cryo-electron microscopy (EM) studies of the FimD usher (a close homolog of PapC) have shown that the usher is a twinned pore in both detergent and lipid bilayers (23, 46). Only one pore is used for secretion, but two pores are required for subunit recruitment (39). For PapC, both monomers and dimers have been described (15, 39). The usher has four functional domains (Fig. ​(Fig.1B):1B): a translocation domain forming a β-barrel with 24 transmembrane β-strands (15, 39), a plug domain in the middle of the translocation domain, and two periplasmic domains, one at each of the N- and C-terminal ends of the usher polypeptide (35, 48). The plug domain has a β-sandwich fold and completely occludes the pore in the inactive usher. Its function, besides gating the channel, seems to be further associated with pilus biogenesis since the deletion of the plug domain abolishes pilus formation in vitro and in vivo (15, 26, 54). The N-terminal domain selectively binds chaperone-subunit complexes (12, 33). The structure of the N-terminal domain of FimD bound to chaperone-subunit complexes indicated that the first 24 residues of FimD are involved in the recognition of chaperone-subunit complexes; the deletion of this region was shown previously to abolish pilus biogenesis (12, 32, 33).The role of the usher C-terminal domain (CTD) is not well understood. The binding of the chaperone-adhesin complex to the usher C terminus was previously demonstrated in vitro (46), while protease susceptibility in FimD shows that, following targeting to the usher N terminus, the chaperone-adhesin complex forms stable interactions with the FimD C terminus, inducing a conformational change in FimD that may be fundamental in the activation step of pilus biogenesis (29, 30, 43). The structure of the C-terminal domain is unknown and is the only part of the CU pilus biogenesis pathway not yet represented in structural terms. Here we provide evidence for the presence of a discrete folding unit in the PapC CTD and report its structure determined by nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography.     

C-terminal Domains of N-Methyl-d-aspartic Acid Receptor Modulate Unitary Channel Conductance and Gating

NMDA receptors (NRs) are glutamate-gated calcium-permeable channels that are essential for normal synaptic transmssion and contribute to neurodegeneration. Tetrameric proteins consist of two obligatory GluN1 (N1) and two GluN2 (N2) subunits, of which GluN2A (2A) and GluN2B (2B) are prevalent in adult brain. The intracellularly located C-terminal domains (CTDs) make a significant portion of mass of the receptors and are essential for plasticity and excitotoxicity, but their functions are incompletely defined. Recent evidence shows that truncation of the N2 CTD alters channel kinetics; however, the mechanism by which this occurs is unclear. Here we recorded activity from individual NRs lacking the CTDs of N1, 2A, or 2B and determined the gating mechanisms of these receptors. Receptors lacking the N1 CTDs had larger unitary conductance and faster deactivation kinetics, receptors lacking the 2A or 2B CTDs had longer openings and longer desensitized intervals, and the first 100 amino acids of the N2 CTD were essential for these changes. In addition, receptors lacking the CTDs of either 2A or 2B maintained isoform-specific kinetic differences and swapping CTDs between 2A and 2B had no effect on single-channel properties. Based on these results, we suggest that perturbations in the CTD can modify the NR-mediated signal in a subunit-dependent manner, in 2A these effects are most likely mediated by membrane-proximal residues, and the isoform-specific biophysical properties conferred by 2A and 2B are CTD-independent. The kinetic mechanisms we developed afford a quantitative approach to understanding how the intracellular domains of NR subunits can modulate the responses of the receptor.     

Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains

Eukaryotic P-type plasma membrane H⁺-ATPases are primary active transport systems that are regulated at the post-translation level by cis-acting autoinhibitory domains, which can be relieved by protein kinase-mediated phosphorylation or binding of specific lipid species. Here we show that lysophospholipids specifically activate a plant plasma membrane H⁺-ATPase (Arabidopsis thaliana AHA2) by a mechanism that involves both cytoplasmic terminal domains of AHA2, whereas they have no effect on the fungal counterpart (Saccharomyces cerevisiae Pma1p). The activation was dependent on the glycerol backbone of the lysophospholipid and increased with acyl chain length, whereas the headgroup had little effect on activation. Activation of the plant pump by lysophospholipids did not involve the penultimate residue, Thr-947, which is known to be phosphorylated as part of a binding site for activating 14-3-3 protein, but was critically dependent on a single autoinhibitory residue (Leu-919) upstream of the C-terminal cytoplasmic domain in AHA2. A corresponding residue is absent in the fungal counterpart. These data indicate that plant plasma membrane H⁺-ATPases evolved as specific receptors for lysophospholipids and support the hypothesis that lysophospholipids are important plant signaling molecules.     

N- and C-terminal modifications of negamycin

Negamycin 1 is a bactericidal antibiotic with activity against Gram-negative bacteria, and served as a template in an antibiotic discovery program. An orthogonally protected beta-amino acid derivative 3a was synthesized and used in parallel synthesis of negamycin derivatives on solid support. This advanced intermediate was also used for N- and C-terminal modifications using solution-phase methodologies. The N-terminal modifications have resulted in the identification of active analogues, whereas the C-terminal modifications resulted in complete loss of antibacterial activity. The N-methyl negamycin analogue, 19a, inhibits protein synthesis (IC(50)=2.3 microM), has antibacterial activity (Escherichia coli, MIC=16 microgram/mL), and is efficacious in an E. coli murine septicemia model (ED(50)=16.3mg/kg).     

The N- and C-terminal Domains of Tomosyn Play Distinct Roles in Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Binding and Fusion Regulation

Tomosyn negatively regulates SNARE-dependent exocytic pathways including insulin secretion, GLUT4 exocytosis, and neurotransmitter release. The molecular mechanism of tomosyn, however, has not been fully elucidated. Here, we reconstituted SNARE-dependent fusion reactions in vitro to recapitulate the tomosyn-regulated exocytic pathways. We then expressed and purified active full-length tomosyn and examined how it regulates the reconstituted SNARE-dependent fusion reactions. Using these defined fusion assays, we demonstrated that tomosyn negatively regulates SNARE-mediated membrane fusion by inhibiting the assembly of the ternary SNARE complex. Tomosyn recognizes the t-SNARE complex and prevents its pairing with the v-SNARE, therefore arresting the fusion reaction at a pre-docking stage. The inhibitory function of tomosyn is mediated by its C-terminal domain (CTD) that contains an R-SNARE-like motif, confirming previous studies carried out using truncated tomosyn fragments. Interestingly, the N-terminal domain (NTD) of tomosyn is critical (but not sufficient) to the binding of tomosyn to the syntaxin monomer, indicating that full-length tomosyn possesses unique features not found in the widely studied CTD fragment. Finally, we showed that the inhibitory function of tomosyn is dominant over the stimulatory activity of the Sec1/Munc18 protein in fusion. We suggest that tomosyn uses its CTD to arrest SNARE-dependent fusion reactions, whereas its NTD is required for the recruitment of tomosyn to vesicle fusion sites through syntaxin interaction.     

Unraveling the Role of the C-terminal Helix Turn Helix of the Coat-binding Domain of Bacteriophage P22 Scaffolding Protein

Many viruses encode scaffolding and coat proteins that co-assemble to form procapsids, which are transient precursor structures leading to progeny virions. In bacteriophage P22, the association of scaffolding and coat proteins is mediated mainly by ionic interactions. The coat protein-binding domain of scaffolding protein is a helix turn helix structure near the C terminus with a high number of charged surface residues. Residues Arg-293 and Lys-296 are particularly important for coat protein binding. The two helices contact each other through hydrophobic side chains. In this study, substitution of the residues of the interface between the helices, and the residues in the β-turn, by aspartic acid was used examine the importance of the conformation of the domain in coat binding. These replacements strongly affected the ability of the scaffolding protein to interact with coat protein. The severity of the defect in the association of scaffolding protein to coat protein was dependent on location, with substitutions at residues in the turn and helix 2 causing the most significant effects. Substituting aspartic acid for hydrophobic interface residues dramatically perturbs the stability of the structure, but similar substitutions in the turn had much less effect on the integrity of this domain, as determined by circular dichroism. We propose that the binding of scaffolding protein to coat protein is dependent on angle of the β-turn and the orientation of the charged surface on helix 2. Surprisingly, formation of the highly complex procapsid structure depends on a relatively simple interaction.     

Effect of mutations of N- and C-terminal charged residues on the activity of LCAT

On the basis of structural homology calculations, we previously showed that lecithin:cholesterol acyltransferase (LCAT), like lipases, belongs to the alpha/beta hydrolase fold family. As there is higher sequence conservation in the N-terminal region of LCAT, we investigated the contribution of the N- and C-terminal conserved basic residues to the catalytic activity of this enzyme. Most basic, and some acidic residues, conserved among LCAT proteins from different species, were mutated in the N-terminal (residues 1;-210) and C-terminal (residues 211;-416) regions of LCAT. Measurements of LCAT-specific activity on a monomeric substrate, on low density lipoprotein (LDL), and on reconstituted high density lipoprotein (rHDL) showed that mutations of N-terminal conserved basic residues affect LCAT activity more than those in the C-terminal region. This agrees with the highest conservation of the alpha/beta hydrolase fold and structural homology with pancreatic lipase observed for the N-terminal region, and with the location of most of the natural mutants reported for human LCAT. The structural homology between LCAT and pancreatic lipase further suggests that residues R80, R147, and D145 of LCAT might correspond to residues R37, K107, and D105 of pancreatic lipase, which form the salt bridges D105-K107 and D105-R37. Natural and engineered mutations at residues R80, D145, and R147 of LCAT are accompanied by a substantial decrease or loss of activity, suggesting that salt bridges between these residues might contribute to the structural stability of the enzyme.     

Synergistic and antagonistic roles of the Sonic hedgehog N- and C-terminal lipids

The Shh protein contains both N-terminal and C-terminal lipids. The functional redundancy of these lipid moieties is presently unclear. Here, we compare the relative roles of the N- and C-terminal lipids in early rat striatal neuronal differentiation, membrane association and multimerization, and ventralizing activity in the zebrafish forebrain. We show that these lipid act synergistically in cell tethering and the formation of a large (L) multimer (669 kDa). However, the C-terminal lipid antagonizes the rat striatal neuronal differentiation-inducing activity of the N-terminal lipid. In addition, multimerization is required but not sufficient for the differentiation-inducing activity. Based on the presence of different N- and C-lipid-containing Shh proteins in the rat embryo, and on their different activities, we propose that both N- and C-terminal lipids are required for the formation of multimers involved in long-range signaling, and that the C-terminal lipid may function in long-range signaling by reducing Shh activity until it reaches its long-range target. Comparative analysis of the ventralizing activities of different N- and C-terminal lipid-containing Shh proteins in the zebrafish forebrain shows that the presence of at least one lipid is required for signaling activity, suggesting that lipid modification of Shh is a conserved requirement for signaling in the forebrain of rodents and zebrafish.     

Probing the Structural and Functional Domains of the CFTR Chloride Channel

The cystic fibrosis transmembrane conductance regulator (CFTR) forms an anion-selective channel involved in epithelial chloride transport. Recent studies have provided new insights into the structural determinants of the channel's functional properties, such as anion selectivity, single-channel conductance, and gating. Using the scanning-cysteine-accessibility method we identified 7 residues in the M1 membrane-spanning segment and 11 residues in and flanking the M6 segment that are exposed on the water-accessible surface of the protein; many of these residues may line the ion-conducting pathway. The pattern of the accessible residues suggests that these segments have a largely -helical secondary structure with one face exposed in the channel lumen. Our results suggest that the residues at the cytoplasmic end of the M6 segment loop back into the channel, narrowing the lumen, and thereby forming both the major resistance to ion movement and the charge-selectivity filter.     

Oligomerization of the Polycystin-2 C-terminal Tail and Effects on Its Ca2+-binding Properties

Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca²⁺-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca²⁺-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca²⁺ bound, although TRP channels are believed to be tetramers. We found that there is only one Ca²⁺-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca²⁺ binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca²⁺ binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca²⁺-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca²⁺ binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca²⁺-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca²⁺-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca²⁺-binding proteins.     

Influence of the N- and C-terminal regions of leu-lys rich antimicrobial peptide on antimicrobial activity

P5 (KWKKLLKKPLLKKLLKKL-NH(2)) is an antibacterial 18-mer Leu-Lys rich peptide from CA (1-8)-MA (1-12) hybrid peptide (CA-MA). Here we show that decreasing the net hydrophobicity and charge of CA-MA by deleting Leu- or Lys- of the N- or C-terminal regions of P5 (P10 or P11). The antimicrobial activity of the peptides was measured by their growth inhibitory effect upon S. aureus, B. subtilis, P. aeruginosa, S. typhimurium, E. coli, T. beigelii and C. albicans. Antimicrobial activity required a full length C-terminus. Confocal microscopy showed that P11 was located in the plasma membrane. In this study, P11, K(3)K(4)L(5)L(6)-deleted peptide, acted independent on the ionic environment. Furthermore, P11 causes significant morphological alterations of the fungal surfaces as shown by scanning electron microscopy.     

Expression and characterization of the N- and C-terminal ATP-binding domains of MRP1

The His(6)-tagged N- and C-terminal nucleotide binding (ATP Binding Cassette, ABC) domains of the human multidrug resistance associated protein, MRP1, were expressed in bacteria in fusion to the bacterial maltose binding protein and a two-step affinity purification was utilized. Binding of a fluorescent ATP-analogue occurred with micromolar dissociation constants, MgATP was able to inhibit the ATP-analogue binding with 70 and 200 micromolar apparent inhibition constants, while AMP was nearly ineffective. Both MRP1 nucleotide binding domains showed ATPase activities (V(max) values between 5-10 nmoles/mg protein/min), which is fifty to hundred times lower than that of parent transporter. The K(M) value of the ATP hydrolysis by the nucleotide binding domains were 1.5 mM and 1.8 mM, which is similar to the K(M) value of the native or the purified and reconstituted transporter, N-ethylmaleinimide and A1F(4) inhibited the ATPase activity of both nucleotide binding domains.     

The Roles of Pore Ring and Plug in the SecY Protein-conducting Channel

The protein-conducting channel, or translocon, is an evolutionarily conserved complex that allows nascent proteins to cross a cellular membrane or integrate into it. The crystal structure of an archaeal translocon, the SecY complex, revealed that two elements contribute to sealing the channel: a small “plug” domain blocking the periplasmic region of the channel, and a pore ring composed of six hydrophobic residues acting as a constriction point at the channel's center. To determine the independent functions of these two elements, we have performed molecular dynamics simulations of the native channel as well as of two recently structurally resolved mutants in which portions of their plugs were deleted. We find that in the mutants, the instability in the plug region leads to a concomitant increase in flexibility of the pore ring. The instability is quantified by the rate of water permeation in each system as well as by the force required for oligopeptide translocation. Through a novel simulation in which the interactions between the plug and water were independently controlled, we find that the role of the plug in stabilizing the pore ring is significantly more important than its role as a purely steric barrier.     

Comparative studies on the functional roles of N- and C-terminal regions of molluskan and vertebrate troponin-I

Vertebrate troponin regulates muscle contraction through alternative binding of the C-terminal region of the inhibitory subunit, troponin-I (TnI), to actin or troponin-C (TnC) in a Ca(2+)-dependent manner. To elucidate the molecular mechanisms of this regulation by molluskan troponin, we compared the functional properties of the recombinant fragments of Akazara scallop TnI and rabbit fast skeletal TnI. The C-terminal fragment of Akazara scallop TnI (ATnI(232-292)), which contains the inhibitory region (residues 104-115 of rabbit TnI) and the regulatory TnC-binding site (residues 116-131), bound actin-tropomyosin and inhibited actomyosin-tropomyosin Mg-ATPase. However, it did not interact with TnC, even in the presence of Ca(2+). These results indicated that the mechanism involved in the alternative binding of this region was not observed in molluskan troponin. On the other hand, ATnI(130-252), which contains the structural TnC-binding site (residues 1-30 of rabbit TnI) and the inhibitory region, bound strongly to both actin and TnC. Moreover, the ternary complex consisting of this fragment, troponin-T, and TnC activated the ATPase in a Ca(2+)-dependent manner almost as effectively as intact Akazara scallop troponin. Therefore, Akazara scallop troponin regulates the contraction through the activating mechanisms that involve the region spanning from the structural TnC-binding site to the inhibitory region of TnI. Together with the observation that corresponding rabbit TnI-fragment (RTnI(1-116)) shows similar activating effects, these findings suggest the importance of the TnI N-terminal region not only for maintaining the structural integrity of troponin complex but also for Ca(2+)-dependent activation.     

Determination of N- and C-terminal borders of the transmembrane domain of integrin subunits

Previous studies on the membrane-cytoplasm interphase of human integrin subunits have shown that a conserved lysine in subunits alpha(2), alpha(5), beta(1), and beta(2) is embedded in the plasma membrane in the absence of interacting proteins (Armulik, A., Nilsson, I., von Heijne, G., and Johansson, S. (1999) in J. Biol. Chem. 274, 37030-37034). Using a glycosylation mapping technique, we here show that alpha(10) and beta(8), two subunits that deviate significantly from the integrin consensus sequences in the membrane-proximal region, were found to have the conserved lysine at a similar position in the lipid bilayer. Thus, this organization at the C-terminal end of the transmembrane (TM) domain seems likely to be general for all 24 integrin subunits. Furthermore, we have determined the N-terminal border of the TM domains of the alpha(2), alpha(5), alpha(10), beta(1), and beta(8) subunits. The TM domain of subunit beta(8) is found to be 22 amino acids long, with a second basic residue (Arg(684)) positioned just inside the membrane at the exoplasmic side, whereas the lipidembedded domains of the other subunits are longer, varying from 25 (alpha(2)) to 29 amino acids (alpha(10)). These numbers implicate that the TM region of the analyzed integrins (except beta(8)) would be tilted or bent in the membrane. Integrin signaling by transmembrane conformational change may involve alteration of the position of the segment adjacent to the conserved lysine. To test the proposed "piston" model for signaling, we forced this region at the C-terminal end of the alpha(5) and beta(1) TM domains out of the membrane into the cytosol by replacing Lys-Leu with Lys-Lys. The mutation was found to not alter the position of the N-terminal end of the TM domain in the membrane, indicating that the TM domain is not moving as a piston. Instead the shift results in a shorter and therefore less tilted or bent TM alpha-helix.