Putative Hinge Region Research Articles

Structural and dynamic insights into the role of conformational switching in the nuclease activity of the Xanthomonas albilineans Cas2 in CRISPR-mediated adaptive immunity.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute a microbial, adaptive immune system countering invading nucleic acids. Cas2 is a universal Cas protein found in all types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition into CRISPR loci. In subtype I-C CRISPR-Cas systems, Cas2 proteins are metal-dependent double-stranded DNA (dsDNA) nucleases, and a pH-dependent conformational transition has been proposed as a prerequisite for catalytic action. Here, we report the crystal structure of Xanthomonas albilineans Cas2 (XaCas2) and provide experimental evidence of a pH-dependent conformational change during functional activation. XaCas2 crystallized at an acidic pH represented a catalytically inactive conformational state in which two Asp8 residues were too far apart to coordinate a single catalytic metal ion. Consistently, XaCas2 exhibited dsDNA nuclease activity only under neutral and basic conditions. Despite the overall structural similarity of the two protomers, significant conformational heterogeneity was evident in the putative hinge regions, suggesting that XaCas2 engages in hinge-bending conformational switching. The presence of a Trp residue in the hinge region enabled the investigation of hinge dynamics by fluorescence spectroscopy. The pH dependence of the fluorescence intensity overlapped precisely with that of nuclease activity. Mutational analyses further suggested that conformational activation proceeded via a rigid-body hinge-bending motion as both D8E and hinge mutations significantly reduced nuclease activity. Together, our results reveal strong correlations between the conformational states, catalytic activity, and hinge dynamics of XaCas2, and provide structural and dynamic insights into the conformational activation of the nuclease function of Cas2.

Crystal Structures of Streptococcus Pyogenes Cas2 Protein at Various pH Conditions

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (cas) proteins provide RNA-mediated adaptive immunity against foreign invading nucleic acids such as phages and plasmids in archaea and bacteria. Cas2 protein is one of the two core Cas proteins are present in all types of CRISPR-Cas systems and is required for new spacer integration into CRISPR loci. Cas2 homologues from several CRISPR-Cas subtypes were characterized as metal-dependent nucleases with different substrate preferences, and it was proposed that a pH-dependent conformational change mediates metal binding and catalysis. Here, we report the X-ray crystal structures of Streptococcus pyogenes Cas2 protein at three different pHs (5.6, 6.5, and 7.5), and the results of its nuclease activity assay at varying pHs (6.0-9.0). While S. pyogenes Cas2 exhibited strongly pH-dependent catalytic activity, there was no significant conformational difference among the three crystal structures. However, structural comparisons with other Cas2 homologues suggest structural variability and the flexible nature of its putative hinge regions, supporting the supposition that conformational change is important for catalysis.

Structural and functional characterization of Streptococcus pyogenes Cas2 protein under different pH conditions

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute an RNA-guided microbial defense system against invading foreign genetic materials. Cas2 is one of the core Cas proteins found universally in all the subtypes of CRISPR-Cas systems and is required for incorporating new spacers into CRISPR loci. Cas2 homologues from different CRISPR-Cas subtypes were characterized previously as metal-dependent nucleases with different substrate preferences, and it was proposed that a pH-dependent conformational change mediates metal binding and catalysis. Here, we report the crystal structures of Streptococcus pyogenes Cas2 at three different pHs (5.6, 6.5, and 7.5), as well as the results of its nuclease activity assay against double-stranded DNAs at varying pHs (6.0–9.0). Although S. pyogenes Cas2 exhibited strongly pH-dependent catalytic activity, there was no significant conformational difference among the three crystal structures. However, structural comparisons with other Cas2 homologues revealed structural variability and the flexible nature of its putative hinge regions, supporting the hypothesis that conformational switching is important for catalysis. Taken together, our results confirm that Cas2 proteins have pH-dependent nuclease activity against double-stranded DNAs, and provide indirect structural evidence for their conformational changes.

Structural and Functional Characterization of the TNT1 Domain of Cardiac Troponin T

Familial Hypertrophic Cardiomyopathy (FHC) is a primary cardiac muscle disorder and a common cause of sudden cardiac death among young people in the field. The majority of disease-causing mutations in the thin filament protein hcTnT are found within the TNT1 domain. This domain has not been crystallized and its structural details are poorly defined, limiting our ability to understand the mechanism of disease for these mutations. A highly charged region is found at the C-terminal end of TNT1 (158-RREEEENRR-166) in which this highly alpha helical domain may unwind to create a flexible hinge that is necessary for normal function. We aim to determine the structural details and function of this region using SDSL-EPR and regulated in vitro motility (R-IVM) assays. The purpose of our R-IVM experiments is two-fold: to functionally analyze our spin labeled proteins and to gain insight into the function of TNT1 in the presence of cysteine substitutions. R-IVM data shows a progressive increase in the severity of the functional effects of cysteine substitution and spin labeling across the putative hinge region, suggesting that this region is dynamically important and may be making critical interactions with other components of the sarcomere. CW-EPR spectra of spin labeled hcTnT in Troponin ternary complexes show an increase in spin label mobility from residue 153 to 157 and 177, consistent with a decrease in alpha helical character across the putative hinge region. Preliminary doubly labeled CW-EPR experiments show that interspin distance between hcTnT residues 157 and 177 exceed 25A. Interspin distance measurements using doubly labeled hcTnT will further elucidate the secondary and tertiary structure of this region. Additional spin label pairs are currently being investigated using both CW-EPR and DEER techniques to determine the structural details of this important region.

Functional and Structural Changes Induced By cTNT-Related FHC Mutations in TNT1 Alter Actomyosin Binding Interactions

Familial Hypertrophic Cardiomyopathy (FHC) is a primary disease of the cardiac sarcomere. Many FHC mutations in hcTnT are found within the TNT1 domain, with a mutational hotpot at residues 160-163. These residues fall within a highly charged region (158-RREEEENRR-166), which may create a flexible hinge necessary for function, the structure and function of which is affected by FHC mutations. We are investigating the effects of these hotspot mutations using in vitro motility (IVM) assays, SDSL-EPR, and transgenic mouse models. IVM data indicate that mutations Δ160E and E163R disrupt actin binding to heavy meromyosin under standard assay conditions. By reducing the ionic strength of the motility solutions, thin filament binding and sliding are restored suggesting that mutations in this region cause disease by disrupting the weak electrostatic interactions between the thin filament and myosin necessary for crossbridge formation. CW-EPR spectra show an increase in spin label isotropic rotational rate at hcTnT residue 153 (upstream of the putative hinge region) between Troponin ternary complexes containing Δ160E verses WT hcTnT, suggesting an increase in flexibility due to backbone changes induced by the mutation. We are expanding our SDSL-EPR experiments with additional cysteine substitutions superimposed onto 160-163 mutant proteins to provide further data regarding secondary structural changes imposed by these mutations. These results correspond with our Δ160E mouse model showing dose dependant myofilament disarray. Preliminary observations of an E163R model suggest that this mutant is less severely affected, tolerating a higher transgene dose. The structural and functional changes observed in vitro may contribute to the structural impairment seen in vivo. By correlating our IVM and SDSL-EPR findings with in vivo data generated from the Δ160E and E163R models, a mechanism of disease for these hotspot mutations can be determined.

Structural and Functional Characterization of cTnT in Familial Hypertrophic Cardiomyopathy

Familial Hypertrophic Cardiomyopathy (FHC) is a primary disease of the cardiac sarcomere. Many disease-causing mutations in the thin filament protein cTnT are found within the TNT1 region. Residues 160-163 represent a mutational hotspot within a highly charged region (158-RREEEENRR-166). In this region, this highly alpha helical domain may unwind to create a flexible hinge that is necessary for function, the structure and dynamics of which may be affected by FHC mutations. We are investigating the structure and function of this region using in vitro motility (IVM) assays and SDSL-EPR. The purpose of our IVM experiments is two-fold: to functionally analyze our spin labeled proteins and to gain insight into the function of TNT1 in the presence of cysteine substitutions and FHC mutations. Preliminary IVM data shows a progressive increase in the severity of the functional effects of cysteine substitution and spin labeling across the putative hinge region (153<168<172), suggesting that this region is dynamically important and may be making critical interactions with other components of the sarcomere. Preliminary CW-EPR spectra show an increase in isotropic rotational rate at residue 153 (upstream of the putative hinge region) between cTnT alone and in the troponin ternary complex, suggesting that there is a decrease in alpha helical character at this residue in the ternary complex. Introduction of Δ160E further increases the isotropic rotational rate, suggesting an increase in flexibility due to backbone changes induced by the mutation. To further investigate structural and functional changes caused by FHC mutations within the putative hinge region, we will continue to expand our IVM functional analyses with additional cysteine substitutions, as well as FHC mutations at residues 160 and 163. Double label SDSL-EPR are currently underway that will provide secondary and tertiary structural information.

Insights into a putative hinge region in elastin using molecular dynamics simulations

The resiliency and elasticity of vertebrate tissues are traced to elastin, a crosslinked protein with extensive hydrophobic regions. There is little discussion in the literature on the structure and dynamics of the alanine-rich crosslinking regions of elastin that comprise a significant part of the native protein. In particular, the region encoded by exons 21 and 23, a contiguous splice form found in all types of human elastin, is believed to be strategically positioned for proper function of the protein, namely, in the reversible elongation and contraction of tissue. Hence, molecular dynamics (MD) calculations on the EX21/23 domain are reported here. This crosslinking domain has been assumed to adopt an architecture in which the putative hinge region links two α-helices. In this paper, we use a homology-based approach to obtain starting structures in the hinge region. The subsequent MD brings new insights into the possibility of fluctuations between “open” and “closed” states, as well as distinguishing structural features of the latter. The significance of these findings towards an enhanced understanding of structure–function relationships in elastin and the elastic fiber is discussed.

Single amino acid sequence polymorphisms in rat cardiac troponin revealed by top-down tandem mass spectrometry.

Heterotrimeric cardiac troponin (cTn) is a critical component of the thin filament regulatory complex in cardiac muscle. Two of the three subunits, cTnI and cTnT, are subject to post-translational modifications such as proteolysis and phosphorylation, but linking modification patterns to function remains a major challenge. To obtain a global view of the biochemical state of cTn in native tissue, we performed high resolution top-down mass spectrometry of cTn heterotrimers from healthy adult rat hearts. cTn heterotrimers were affinity purified, desalted and then directly subjected to mass spectrometry using a 7 Tesla Thermo LTQ-FT-ICR instrument equipped with an ESI source. Molecular ions for N-terminally processed and acetylated cTnI and cTnT were readily detected as were other post-translationally modified forms of these proteins. cTnI was phosphorylated with a distribution of un-, mono- and bisphosphorylated forms of 41 +/- 3%, 46 +/- 1%, 13 +/- 3%, respectively. cTnT was predominantly monophosphorylated and partially proteolyzed at the Glu(29)-Pro(30) peptide bond. Also observed in high resolution spectra were 'shadow' peaks of similar intensity to 'parent' peaks exhibiting masses of cTnI+16 Da and cTnT+128 Da, subsequently shown by tandem mass spectrometry (MS/MS) to be single amino acid polymorphisms. Intact and protease-digested cTn subunits were fragmented by electron capture dissociation or collision activated dissociation to localize an Ala/Ser polymorphism at residue 7 of cTnI. Similar analysis of cTnT localized an additional Gln within a three residue alternative splice site beginning at residue 192. Besides being able to provide unique insights into the global state of post-translational modification of cTn subunits, high resolution top-down mass spectrometry readily revealed naturally occurring single amino acid sequence variants including a genetic polymorphism at residue 7 in cTnI, and an alternative splice isoform that affects a putative hinge region around residue 192 of cTnT, all of which co-exist within a single rat heart.

A high resolution structure of the putative hinge region in M2 channel-lining segments of the nicotinic acetylcholine receptor

Transmembrane peptide helices play key roles in signal transduction across cell membranes, yet little is known about their high-resolution structure or the role membrane composition plays in their association, structure, dynamics and ultimately their performance. Using magic angle spinning (MAS) homonuclear dipolar recoupling experiments, the backbone structure at positions L10, L11, and A12 of the M2 ion channel peptide was determined in two lipid systems. Their measurements are in agreement with M2 forming transmembrane helices, but the torsion angles vary considerably from common α−helical values. These measurements show remarkable agreement with a previous computational model of M2 peptides forming a pore domain in which their helices are kinked near the central leucine, L11 [R. Sankararamakrishnan, C. Adcock, M.S.P. Sansom, The pore domain of the nicotinic acetylcholine receptor: Molecular modeling, pore dimensions, and electrostatics, Biophys. J. 71 (1996) 1659–1671]. The generation of high resolution data for transmembrane helices is of critical importance in refining structures for membrane protein and developing models of helix packing interactions.

Structural Determinants of Cross-linking and Hydrophobic Domains for Self-Assembly of Elastin-like Polypeptides

Elastin is a major structural protein found in large blood vessels, lung, ligaments, and skin, imparting the physical properties of extensibility and elastic recoil to these tissues. To achieve the required structural durability of the elastic matrix, the elastin monomer, tropoelastin, undergoes ordered assembly into a covalently cross-linked, fibrillar polymeric structure. Human tropoelastin consists of 34 exons coding for alternating hydrophobic and cross-linking domains. Using a series of well-defined recombinant polypeptides based on human elastin sequences mimicking native elastin, we have previously investigated the role of sequence and context of hydrophobic domains in elastin self-assembly. Here, we demonstrate that the structure of both cross-linking and hydrophobic domains have significant effects on the assembly of these polypeptides. Removing a putative flexible hinge region in the center of a cross-linking domain substantially increased the alpha-helical content and strongly promoted their self-aggregation. However, while trifluoroethanol (TFE) promoted and urea inhibited self-assembly of these polypeptides, these effects were not predominantly due to altered alpha-helicity of the polypeptides. Our results suggest that, while increased alpha helicity also favors this process, the major effect of TFE to promote organized self-assembly of elastin-like polypeptides is likely related to direct effects of this cosolvent on hydrophobic domains. Such simple elastin polypeptide models can provide an important tool for understanding the relationships between sequence, structure, and polymeric assembly of elastin.

The Crystal Structure of the Plexin-Semaphorin-Integrin Domain/Hybrid Domain/I-EGF1 Segment from the Human Integrin β2 Subunit at 1.8-Å Resolution

Integrins are modular (alphabeta) heterodimeric proteins that mediate cell adhesion and convey signals across the plasma membrane. Interdomain motions play a key role in signal transduction by propagating structural changes through the molecule, thus controlling the activation state and adhesive properties of the integrin. We expressed a soluble fragment of the human integrin beta2 subunit comprising the plexin-semaphorin-integrin domain (PSI)/hybrid domain/I-EGF1 fragment and present its crystal structure at 1.8-A resolution. The structure reveals an elongated molecule with a rigid architecture stabilized by nine disulfide bridges. The PSI domain is located centrally and participates in the formation of extended interfaces with the hybrid domain and I-EGF1 domains, respectively. The hybrid domain/PSI interface involves the burial of an Arg residue, and contacts between PSI and I-EGF1 are mainly mediated by well conserved Arg and Trp residues. Conservation of key interacting residues across the various integrin beta subunits sequences suggests that our structure represents a good model for the entire integrin family. Superposition with the integrin beta3 receptor in its bent conformation suggests that an articulation point is present at the linkage between its I-EGF1 and I-EGF2 modules and underlines the importance of this region for the control of integrin-mediated cell adhesion.

Deletion of the cnxE gene encoding the gephyrin-like protein involved in the final stages of molybdenum cofactor biosynthesis in Aspergillus nidulans.

The Aspergillus nidulans cnxE gene, required for molybdenum cofactor biosynthesis, was isolated by functional complementation of an Escherichia coli mogA mutant strain. The deduced CnxE polypeptide consists of two domains which display similarity to the E. coli proteins MoeA and MogA, respectively, separated by a putative hinge region of around 58 amino acid residues which is notably histidine rich. A deletion mutant lacking the entire cnxE gene, including both MoeA-like and MogA-like domains, was identified. Compared to the wild type, a small increase in the intermediate precursor Z was observed in the deletion strain but was significant only under conditions in which the molybdoenzyme nitrate reductase was induced. Elevated levels of the pathway intermediate molybdopterin were found both under nitrate reductase-inducing and non-inducing conditions in the deletion mutant compared to the wild type. This increase is in contrast to previous results for cnxABC, cnxF, cnxG, and cnxH mutants, in which the levels of molybdopterin were substantially reduced, and therefore supports previously published classical genetic and biochemical studies that indicated that the CnxE protein is likely to be involved in the final stages of molybdenum cofactor biosynthesis. We have found no evidence during our chemical analysis for any involvement of this protein in the intermediate section of the molybdenum cofactor biosynthetic pathway (i.e. in the synthesis of molybdopterin from precursor Z), as has been suggested previously for E. coli MoeA. The 2.5-kb cnxE transcript is not abundant and appears to be expressed constitutively.

Cloning and characterization of cDNAs encoding four different canine immunoglobulin γ chains

cDNAs encoding four different canine immunoglobulin G (caIgG) γ chains were identified in this study. One of these IgG γ chain cDNAs, (caIgG-A), represents 92.5% of the IgG γ chain cDNAs in a dog spleen cell cDNA library; a second partial IgG γ chain cDNA (caIgG-B) was also identified in the library. The other two IgG γ chain cDNAs (caIgG-C and caIgG-D) were RT-PCR amplified from canine lymphoma samples. Comparison of the four different canine IgG γ chain cDNAs showed homologies from 83.6 to 89.2% and from 73.1 to 81.8% at nucleotide and amino acid sequence levels, respectively. Despite the high similarity in CH1, CH2 and CH3 domains among the different caIgG γ chains, the hinge regions were distinct, sharing only 19.0–35.2% homology at the amino acid level. No multiple duplication of the hinge region, as reported for human IgG1 and IgG3, was detected in any of the canine IgG γ chains. The numbers of cysteines in the putative hinge regions were found to be 3, 2, 7 and 3 for the four canine IgG heavy γ chains (A, B, C and D), respectively. Specific primers were designed based on caIgG γ chain hinge region DNA sequences and were used in RT-PCR for measuring different caIgG γ chain mRNA levels in canine PBMC samples.

Novel insights into catalytic mechanism from a crystal structure of human topoisomerase I in complex with DNA.

Human topoisomerase I helps to control the level of DNA supercoiling in cells and is vital for numerous DNA metabolic events, including replication, transcription, and recombination. The 2.6 A crystal structure of human topoisomerase I in noncovalent complex with a DNA duplex containing a cytosine at the -1 position of the scissile strand rather than the favored thymine is reported. The hydrogen bond between the O2 position of this -1 base and the epsilon-amino of the conserved Lys-532 residue, the only base-specific contact observed previously in the human topoisomerase I-DNA interaction, is maintained in this complex. Several unique features of this structure, however, have implications for the DNA-binding and active-site mechanisms of the enzyme. First, the ends of the DNA duplex were observed to shift by up to 5.4 A perpendicular to the DNA helical axis relative to structures reported previously, suggesting a novel degree of plasticity in the interaction between human topoisomerase I and its DNA substrate. Second, 12 additional residues at the NH(2) terminus of the protein (Trp-203-Gly-214) could be built in this structure, and they were found to pack against the putative hinge region implicated in the clamping of the enzyme around duplex DNA. Third, a water molecule was observed adjacent to the scissile phosphate and the active-site residues; the potential specific base character of this solvent molecule in the active-site mechanism of the enzyme is discussed. Fourth, the scissile phosphate group was found to be rotated by 75 degrees, bringing Lys-532 into hydrogen-bonding distance of one of the nonbridging phosphate oxygens. This orientation of the scissile phosphate group implicates Lys-532 as a fifth active-site residue, and also mimics the orientation observed for the 3'-phosphotyrosine linkage in the covalent human topoisomerase I-DNA complex structure. The implications of these structural features for the mechanism of the enzyme are discussed, including the potential requirement for a rotation of the scissile phosphate group during DNA strand cleavage and covalent attachment.

Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E

The N-terminal domain of human apolipoprotein E3 (apoE3) adopts an elongated, globular four helix bundle conformation in the lipid-free state. Upon lipid binding, the protein is thought to undergo a significant conformational change that is essential for manifestation of its low density lipoprotein receptor recognition properties. We have used fluorescence resonance energy transfer (FRET) to characterize helix repositioning which accompanies lipid interaction of this protein. ApoE3(1-183) possesses a single cysteine at position 112 and four tryptophan residues (positions 20, 26, 34, and 39). Modification of Cys112 with the chromophore, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)etheylenediamine (AEDANS) was specific and did not alter the secondary structure content of the protein. The efficiency of energy transfer from donor Trp residues to the AEDANS moiety was 49% in buffer, consistent with close proximity of the chromophores. Guanidine HCl titration experiments induced characteristic changes in the efficiency of energy transfer, indicating that FRET data faithfully reports on the conformational status of the protein. Interaction of AEDANS-apoE3(1-183) with dimyristoylphosphatidylcholine to form disk particles, or with detergent micelles, resulted in large decreases in the efficiency of energy transfer. Distance calculations based on the FRET measurements revealed that lipid binding increases the average distance between the four Trp donors and the AEDANS acceptor from 23 A to 44 A. The results obtained demonstrate the utility of FRET to investigate conformational adaptations of exchangeable apolipoproteins and are consistent with the hypothesis that, upon lipid binding, apoE3(1-183) undergoes conformational opening, repositioning helix 1 and 3 to adopt a receptor-active conformation.

Vascular Smooth Muscle Cells Express Multiple Type I Receptors for TGF-β,Activin, and Bone Morphogenetic Proteins

Vascular smooth muscle cells (VSMCs) are known to produce activins, bone morphogenetic proteins and transforming growth factor-βs (TGF-βs). To determine whether these TGF-β superfamily members exert autocrine effects on VSMCs we examined whether specific type I receptors (ALKs) for such peptides were expressed by the cells. RNA from both quiescent or growth-factor-activated VSMCs was reverse transcribed then cDNAs encoding ALK-2, ALK-3, ALK-5, and ALK-6 were amplified and characterised using specific PCR primers. All four ALK mRNAs were abundantly expressed. The ALK-5 fragment harbored a deletion of 12 nucleotides, removing 4 extracellular amino acids (Gly-Pro-Ser-Val) adjacent to its transmembrane domain. This deletion, which may arise from anomalous splicing of heteronuclear RNA, is likely to influence formation of the ALK-5:type II receptor complex through conformational changes associated with removal of a putative hinge region containing proline. Our finding that multiple ALKs are expressed by VSMCs would account for the multiplicity of effects TGF-β peptides exert on these cells.

Dimer/monomer equilibrium and domain separations of Escherichia coli ribosomal protein L7/L12.

The dimer to monomer equilibrium and interdomain separations of cysteine variants of L7/L12 have been investigated using fluorescence spectroscopy. Steady-state polarization measurements on cysteine containing variants of L7/L12, labeled with 5-(iodoacetamido)fluorescein, demonstrated dimer to monomer dissociation constants near 30 nM for variants labeled at position 33, in the N-terminal domain, and positions 63 and 89, in the C-terminal domain. A dissociation constant near 300 nM was determined for a variant labeled at position 12, in the N-terminal domain. The polarization of a labeled C-terminal fragment did not change over the range of 200 microM to 1 nM, indicating that this construct remains monomeric at these concentrations, whereas a dimer to monomer dissociation constant near 300 nM was observed for an FITC labeled N-terminal fragment. Intersubunit fluorescence resonance energy self-transfer was observed when appropriate probes were attached to cysteines at residues 12 or 33, located in the N-terminal domain. Probes attached to cysteines at positions 63 or 89 in the C-terminal domain, however, did not exhibit intersubunit self-transfer. These results indicate that these residues in the C-terminal domains are, on average, separated by greater than 85 A. Intersubunit self-transfer does occur in a C-89 double mutation variant lacking 11 residues in the putative hinge region, indicating that the loss of the hinge region brings the two C-terminal domains closer together. Rapid subunit exchange between unlabeled wild-type L7/L12 and L7/L12 variants labeled in the N-terminal domain was also demonstrated by the loss of self-transfer upon mixing of the two proteins.

Chimeric horse/human recombinant C9 proteins identify the amino acid sequence in horse C9 responsible for restriction of hemolysis.

Equine C9, in contrast to human C9, has extremely low hemolytic activity against most mammalian erythrocytes, although the amino acid sequences of both proteins show 77% identity. In an attempt to define the region of human C9 responsible for conferring its lytic activity, or conversely, the region of equine C9 responsible for its restriction, recombinant human and equine C9 and four chimeric human/equine C9 proteins were constructed and expressed in COS-7 cells. Recombinant human and equine C9 displayed hemolytic profiles similar to those of the purified native proteins. Exchange of a fragment extending from residues 145 to 290 in horse C9 with the corresponding one from human C9 created a fully hemolytic protein. This region contains the putative hinge region but not the membrane-interacting domain. Nonlytic chimeric C9 proteins inhibited hemolysis and binding of human C9 to EAC1-8 cells, indicating that they bind to their receptor, but subsequent unfolding or insertion into the membrane is impaired. These results suggest that restriction factors, such as glycophorin, CD59, or homologous restriction factor, on erythrocytes may limit the activity of horse C9 by interacting with its hinge region. In support of this conclusion direct binding of CD59 to immobilized horse C9 was detected by ligand blotting, and it was observed that a polyclonal anti-CD59 Ab enhanced human and horse C9-mediated hemolysis of human EAC1-7, but the increase in hemolytic activity of horse C9 by inhibition of CD59 was less than what could be achieved by insertion of the human C9 hinge region into horse C9.

The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis

A variant form of Escherichia coli ribosomal protein L7/L12 that lacked residues 42 to 52 (L7/L12 Δ42–52) in the hinge region was shown previously to be completely inactive in supporting polyphenylalanine synthesis although it bound to L7/L12 deficient core particles with the normal stoichiometry of four copies per particle (Oleinikov AV, Perroud B, Wang B, Traut RR (1993) J Biol Chem, 268, 917–922). The result suggested that the hinge confers flexibility that is required for activity because the resulting bent conformation allows the distal C-terminal domain to occupy a location on the body of the large ribosomal subunit proximal to the base of the L7/L12 stalk where elongation factors bind. Factor binding to the hinge-truncated variant was tested. As an alternative strategy to deleting residues from the hinge, seven amino acid residues within the putative hinge region were replaced by seven consecutive proline residues in an attempt to confer increased rigidity that might reduce or eliminate the bending of the molecule inferred to be functionally important. This variant, L7/L12: (Pro) 7, remained fully active in protein synthesis. Whereas the binding of both factors in ribosomes containing L7/L12:Δ42–52 was decreased by about 50%, there was no loss of factor binding in ribosomes containing L7/L12:(Pro) 7, as predicted from the retention of protein synthesis activity. The factor:ribosome complexes that contained L7/L12:Δ42–52 had the same low level of GTP hydrolysis as the core particles completely lacking L7/L12 and EF-G did not support translocation measured by the reaction of phe-tRNA bounds in hr Asite with puromycin. It is concluded that the hinge region is required for the functionally productive binding of elongation factors, and the defect in protein synthesis reported previously is due to this defect. The variant produced by the introduction of the putative rigid Pro 7 sequence retains sufficient flexibility for full activity.

A synthetic peptide from complement protein C9 binds to CD59 and enhances lysis of human erythrocytes by C5b-9.

The membrane glycoprotein CD59 protects host cells from homologous complement attack by inhibiting the assembly of the membrane attack complex. CD59 binds to C8 and C9 in the nascent membrane attack complex and interferes with C9 membrane insertion and polymerization. We show here that a synthetic peptide from the putative C9 hinge region, postulated to be involved in the rearrangement of C9 globular domains during membrane insertion, binds specifically to CD59 and enhances lysis of human erythrocytes by the terminal complement C5b-9 complex. The peptide, C9H, caused a dose-dependent increase in the sensitivity of human erythrocytes to C5b-9-mediated lysis by interfering with the final C9 binding and/or membrane insertion step. C9H exhibited species-specificity, since it had no activity against guinea pig C8 and C9 or on the putative functional homologues of CD59 in guinea pig erythrocytes. A direct association between CD59 and C9H was suggested by two different binding experiments: C9H inhibited the binding of 125I-labeled CD59 to immobilized C9, and C9H immobilized to microtiter plates bound purified CD59 and selectively recognized CD59 from extracts of detergent-solubilized human erythrocyte membranes. These data indicate that the peptide C9H corresponds to a region of the CD59 binding site of C9.