Insights into dimerization and four-helix bundle formation found by dissection of the dimer interface of the GrpE protein from Escherichia coli

The GrpE heat shock protein from Escherichia coli has a homodimeric structure. The dimer interface encompasses two long alpha-helices at the NH(2)-terminal end from each monomer (forming a "tail"), which lead into a small four-helix bundle from which each monomer contributes two short sequential alpha-helices in an antiparallel topological arrangement. We have created a number of different deletion mutants of GrpE that have portions of the dimer interface to investigate requirements for dimerization and to study four-helix bundle formation. Using chemical crosslinking and analytical ultracentrifugation techniques to probe for multimeric states, we find that a mutant containing only the long alpha-helical tail portion (GrpE1-88) is unable to form a dimer, most likely due to a decrease in alpha-helical content as determined by circular dichroism spectroscopy, thus one reason for a dimeric structure for the GrpE protein is to support the tail region. Mutants containing both of the short alpha-helices (GrpE1-138 and GrpE88-197) are able to form a dimer and presumably the four-helix bundle at the dimer interface. These two mutants have equilibrium constants for the monomer-dimer equilibrium that are very similar to the full-length protein suggesting that the tail region does not contribute significantly to the stability of the dimer. Interestingly, one mutant that contains just one of the short alpha-helices (GrpE1-112) exists as a tetrameric species, which presumably is forming a four-helix bundle structure. A proposed model is discussed for this mutant and its relevance for factors influencing four-helix bundle formation.     

Deletion of DnaK's lid strengthens binding to the nucleotide exchange factor,GrpE: a kinetic and thermodynamic analysis

In this study, we have used surface plasmon resonance (SPR) and isothermal microtitration calorimetry (ITC) to study the mechanism of complex formation between the Hsp70 molecular chaperone, DnaK, and its cochaperone, GrpE, which is a nucleotide exchange factor. Experiments were geared toward understanding the influence of DnaK's three domains, the ATPase (residues 1-388), substrate-binding (residues 393-507), and lid (residues 508-638) domains, on complex formation with GrpE. We show that the equilibrium dissociation constants for the interaction of GrpE with wtDnaK, lidless DnaK(2-517), the ATPase domain (2-388), and the substrate-binding fragment (393-507) are 64 (+/-16) nM, 4.0 (+/-1.5) nM, 35 (+/-10) nM, and 67 (+/-11) microM, respectively, and that the on-rate constant for the different reactions varies by over 4 orders of magnitude. SPR experiments revealed that GrpE-DnaK(393-507) complex formation is inhibited by added peptide and abolished when the 33-residue flexible "tail" of GrpE is deleted. Such results strongly suggest that the 33-residue flexible N-terminal tail of GrpE binds in the substrate-binding pocket of DnaK. This unique mode of binding between GrpE's tail and DnaK contributes to, but does not fully explain, the decrease in K(d) from 64 to 4 nM upon deletion of DnaK's lid. The possibility that deletion of DnaK's lid creates a more symmetrically shaped molecule, with enhanced affinity to GrpE, is also discussed. Our results reveal a complex set of molecular interactions between DnaK and its cochaperone GrpE. We discuss the impact of each domain on complex formation and dissociation.     

GrpE N-terminal domain contributes to the interaction with Dnak and modulates the dynamics of the chaperone substrate binding domain

GrpE acts as a nucleotide exchange factor for DnaK, the main Hsp70 protein in bacteria, accelerating ADP/ATP exchange by several orders of magnitude. GrpE is a homodimer, each subunit containing three structural domains: a N-terminal unordered segment, two long coils and a C-terminal globular domain formed by a four-helix bundle, and a β-subdomain. GrpE association to DnaK nucleotide-binding domain involves side-chain and backbone interactions located within the “headpiece” of the cochaperone, which consists of the C-terminal half of the coils, the four-helix bundle and the β-subdomain. However, the role of the GrpE N-terminal region in the interaction with DnaK and the activity of the cochaperone remain controversial. In this study we explore the contribution of this domain to the binding reaction, using the wild-type proteins, two deletion mutants of GrpE (GrpE_34-197 and GrpE_69-197) and the isolated DnaK nucleotide-binding domain. Analysis of the thermodynamic binding parameters obtained by isothermal titration calorimetry shows that both GrpE N-terminal segments, 1-33 and 34-68, contribute to the binding reaction. Partial proteolysis and substrate dissociation kinetics also suggest that the N-terminal half of GrpE coils (residues 34-68) interacts with DnaK interdomain linker, regulates the nucleotide exchange activity of the cochaperone and is required to stabilize DnaK-substrate complexes in the ADP-bound conformation.     

GrpE, a nucleotide exchange factor for DnaK

The cochaperone GrpE functions as a nucleotide exchange factor to promote dissociation of adenosine 5'-diphosphate (ADP) from the nucleotide-binding cleft of DnaK. GrpE and the DnaJ cochaperone act in concert to control the flux of unfolded polypeptides into and out of the substrate-binding domain of DnaK by regulating the nucleotide-bound state of DnaK. DnaJ stimulates nucleotide hydrolysis, and GrpE promotes the exchange of ADP for adenosine triphosphate (ATP) and also augments peptide release from the DnaK substrate-binding domain in an ATP-independent manner. The eukaryotic cytosol does not contain GrpE per se because GrpE-like function is provided by the BAG1 protein, which acts as a nucleotide exchange factor for cytosolic Hsp70s. GrpE, which plays a prominent role in mitochondria, chloroplasts, and bacterial cytoplasms, is a fascinating molecule with an unusual quaternary structure. The long alpha-helices of GrpE have been hypothesized to act as a thermosensor and to be involved in the decrease in GrpE-dependent nucleotide exchange that is observed in vitro at temperatures relevant to heat shock. This review describes the molecular biology of GrpE and focuses on the structural and kinetic aspects of nucleotide exchange, peptide release, and the thermosensor hypothesis.     

Influence of GrpE on DnaK-substrate interactions

The DnaK chaperone of Escherichia coli assists protein folding by an ATP-dependent interaction with short peptide stretches within substrate polypeptides. This interaction is regulated by the DnaJ and GrpE co-chaperones, which stimulate ATP hydrolysis and nucleotide exchange by DnaK, respectively. Furthermore, GrpE has been claimed to trigger substrate release independent of its role as a nucleotide exchange factor. However, we show here that GrpE can accelerate substrate release from DnaK exclusively in the presence of ATP. In addition, GrpE prevented the association of peptide substrates with DnaK through an activity of its N-terminal 33 amino acids. A ternary complex of GrpE, DnaK, and a peptide substrate could be observed only when the peptide binding to DnaK precedes GrpE binding. Furthermore, we demonstrate that GrpE slows down the release of a protein substrate, sigma(32), from DnaK in the absence of ATP. These findings suggest that the ATP-triggered dissociation of GrpE and substrates from DnaK occurs in a concerted fashion.     

Crystal structure of DnaK protein complexed with nucleotide exchange factor GrpE in DnaK chaperone system: insight into intermolecular communication

The conserved, ATP-dependent bacterial DnaK chaperones process client substrates with the aid of the co-chaperones DnaJ and GrpE. However, in the absence of structural information, how these proteins communicate with each other cannot be fully delineated. For the study reported here, we solved the crystal structure of a full-length Geobacillus kaustophilus HTA426 GrpE homodimer in complex with a nearly full-length G. kaustophilus HTA426 DnaK that contains the interdomain linker (acting as a pseudo-substrate), and the N-terminal nucleotide-binding and C-terminal substrate-binding domains at 4.1-Å resolution. Each complex contains two DnaKs and two GrpEs, which is a stoichiometry that has not been found before. The long N-terminal GrpE α-helices stabilize the linker of DnaK in the complex. Furthermore, interactions between the DnaK substrate-binding domain and the N-terminal disordered region of GrpE may accelerate substrate release from DnaK. These findings provide molecular mechanisms for substrate binding, processing, and release during the Hsp70 chaperone cycle.     

The NH2-terminal domain of the chloroplast GrpE homolog CGE1 is required for dimerization and cochaperone function in vivo

GrpE proteins function as nucleotide exchange factors for DnaK-type Hsp70s. We have previously identified a chloroplast homolog of GrpE in Chlamydomonas reinhardtii, termed CGE1. CGE1 exists as two isoforms, CGE1a and CGE1b, which are generated by temperature-dependent alternative splicing. CGE1b contains additional valine and glutamine residues in its extreme NH2-terminal region. Here we show that CGE1a is predominant at lower temperatures but that CGE1b becomes as abundant as CGE1a at elevated temperatures. Coimmunoprecipitation experiments revealed that CGE1b had a approximately 25% higher affinity for its chloroplast chaperone partner HSP70B than CGE1a. Modeling of the structure of CGE1b revealed that the extended alpha-helix formed by GrpE NH2 termini is 34 amino acids longer in CGE1 than in Escherichia coli GrpE and appears to contain a coiled coil motif. Progressive deletions of this coiled coil increasingly impaired the ability of CGE1 to form dimers, to interact with DnaK at elevated temperatures, and to complement temperature-sensitive growth of a DeltagrpE E. coli strain. In contrast, deletion of the four-helix bundle required for dimerization of E. coli GrpE did not affect CGE1 dimer formation. Circular dichroism measurements revealed that CGE1, like GrpE, undergoes two thermal transitions, the first of which is in the physiologically relevant temperature range (midpoint approximately 45 degrees C). Truncating the NH2-terminal coiled coil shifted the second transition to lower temperatures, whereas removal of the four-helix bundle abolished the first transition. Our data suggest that bacterial GrpE and chloroplast CGE1 share similar structural and biochemical properties, but some of these, like dimerization, are realized by different domains.     

Formation in vitro of complexes between an abnormal fusion protein and the heat shock proteins from Escherichia coli and yeast mitochondria.

Heat shock proteins (HSPs) of the Hsp70 and GroEL families associate with a variety of cell proteins in vivo. However, the formation of such complexes has not been systematically studied. A 31-kDa fusion protein (CRAG), which contains 12 residues of cro repressor, truncated protein A, and 14 residues of beta-galactosidase, when expressed in Escherichia coli, was found in complexes with DnaK, GrpE, protease La, and GroEL. When an E. coli extract not containing CRAG was applied to an affinity column containing CRAG, DnaK, GroEL, and GrpE were selectively bound. These HSPs did not bind to a normal protein A column. DnaK, GrpE, and the fraction of GroEL could be eluted from the CRAG column with ATP but not with a nonhydrolyzable ATP analog. The ATP-dependent release of DnaK and GroEL also required Mg2+, but GrpE dissociated with ATP alone. The binding and release of DnaK and GroEL were independent events, but the binding of GrpE required DnaK. Inactivation of DnaJ, GrpE, and GroES did not affect the association or dissociation of DnaK or GroEL from CRAG. The DnaK and GrpE proteins could be eluted with 10(-6) M ATP, but 10(-4) M was required for GroEL release. This approach allows a one-step purification of these proteins from E. coli and also the isolation of the DnaK and GroEL homologs from yeast mitochondria. Competition experiments with oligopeptide fragments of CRAG showed that DnaK and GroEL interact with different sites on CRAG and that the cro-derived domain of CRAG contains the DnaK-binding site.     

Interaction between the nucleotide exchange factor Mge1 and the mitochondrial Hsp70 Ssc1

Function of Hsp70s such as DnaK of the Escherichia coli cytoplasm and Ssc1 of the mitochondrial matrix of Saccharomyces cerevisiae requires the nucleotide release factors, GrpE and Mge1, respectively. A loop, which protrudes from domain IA of the DnaK ATPase domain, is one of six sites of interaction revealed in the GrpE:DnaK co-crystal structure and has been implicated as a functionally important site in both DnaK and Ssc1. Alanine substitutions for the amino acids (Lys-108 and Arg-213 of Mge1) predicted to interact with the Hsp70 loop were analyzed. Mge1 having both substitutions was able to support growth in the absence of the essential wild-type protein. K108A/R213A Mge1 was able to stimulate nucleotide release from Ssc1 and function in refolding of denatured luciferase, albeit higher concentrations of mutant protein than wild-type protein were required. In vitro and in vivo assays using K108A/R213A Mge1 and Ssc1 indicated that the disruption of contact at this site destabilized the interaction between the two proteins. We propose that the direct interaction between the loop of Ssc1 and Mge1 is not required to effect nucleotide release but plays a role in stabilization of the Mge1-Ssc1 interaction. The robust growth of the K108A/R213A MGE1 mutant suggests that the interaction between Mge1 and Ssc1 is tighter than required for function in vivo.     

Constraints imposed by protease accessibility on the trans-membrane and surface topography of the colicin E1 ion channel.

The surface topography of a 190-residue COOH-terminal colicin E1 channel peptide (NH2-Met 333-Ile 522-COOH) bound to uniformly sized 0.2-micron liposomes was probed by accessibility of the peptide to proteases in order (1) to determine whether the channel structure contains trans-membrane segments in addition to the four alpha-helices previously identified and (2) to discriminate between different topographical possibilities for the surface-bound state. An unfolded surface-bound state is indicated by increased trypsin susceptibility of the bound peptide relative to that of the peptide in aqueous solution. The peptide is bound tightly to the membrane surface with Kd < 10(-7) M. The NH2-terminal 50 residues of the membrane-bound peptide are unbound or loosely bound as indicated by their accessibility to proteases, in contrast with the COOH-terminal 140 residues, which are almost protease inaccessible. The general protease accessibility of the NH2-terminal segment Ala 336-Lys 382 excludes any model for the closed channel state that would include trans-membrane helices on the NH2-terminal side of Lys 382. Lys 381-Lys 382 is a major site for protease cleavage of the surface-bound channel peptide. A site for proteinase K cleavage just upstream of the amphiphilic gating hairpin (K420-K461) implies the presence of a surface-exposed segment in this region. These protease accessibility data indicate that it is unlikely that there are any alpha-helices on the NH2-terminal side of the gating hairpin K420-K461 that are inserted into the membrane in the absence of a membrane potential. A model for the topography of an unfolded monomeric surface-bound intermediate of the colicin channel domain, including a trans-membrane hydrophobic helical hairpin and two or three long surface-bound helices, is proposed.     

Molecular structure of Rarobacter faecitabidus protease I. A yeast-lytic serine protease having mannose-binding activity.

Rarobacter faecitabidus protease I (RPI) is a serine protease exhibiting lytic activity toward living yeast cells. RPI is similar to elastase in its substrate specificity and has a lectin-like affinity for mannose. The gene encoding RPI was cloned to elucidate its structure and function. And its nucleotide sequence revealed that it contains an open reading frame encoding a 525-amino acid protein. Homology comparison indicated that pre-pro-RPI consists of three domains: (1) an NH2-terminal prepro domain not found in the mature form of RPI, (2) a protease domain homologous to the trypsin family of serine proteases, and (3) a COOH-terminal domain homologous to the COOH-terminal part of Oerskovia xanthineolytica beta-1,3-glucanase and the NH2-terminal part of the ricin B chain, a lectin isolated from the part of the ricin B chain, a lectin isolated from the castor bean. The RPI gene and its mutant were subsequently expressed in Escherichia coli under its beta-galactosidase promoter to investigate the function of the COOH-terminal domain. The mutant RPI, whose COOH-terminal domain was truncated by site-directed mutagenesis, lost both its mannose-binding and yeast-lytic activity, although the protease activity was not affected. These findings suggest that the COOH-terminal domain actually participates in the mannose-binding activity and is required for yeast-lytic activity.     

The rod domain of NF-L determines neurofilament architecture, whereas the end domains specify filament assembly and network formation

Neurofilaments, assembled from NF-L, NF-M, and NF-H subunits, are the most abundant structural elements in myelinated axons. Although all three subunits contain a central, alpha-helical rod domain thought to mediate filament assembly, only NF-L self-assembles into 10-nm filaments in vitro. To explore the roles of the central rod, the NH2- terminal head and the COOH-terminal tail domain in filament assembly, full-length, headless, tailless, and rod only fragments of mouse NF-L were expressed in bacteria, purified, and their structure and assembly properties examined by conventional and scanning transmission electron microscopy (TEM and STEM). These experiments revealed that in vitro assembly of NF-L into bona fide 10-nm filaments requires both end domains: whereas the NH2-terminal head domain promotes lateral association of protofilaments into protofibrils and ultimately 10-nm filaments, the COOH-terminal tail domain controls lateral assembly of protofilaments so that it terminates at the 10-nm filament level. Hence, the two end domains of NF-L have antagonistic effects on the lateral association of protofilaments into higher-order structures, with the effect of the COOH-terminal tail domain being dominant over that of the NH2-terminal head domain. Consideration of the 21-nm axial beading commonly observed with 10-nm filaments, the approximate 21-nm axial periodicity measured on paracrystals, and recent cross-linking data combine to support a molecular model for intermediate filament architecture in which the 44-46-nm long dimer rods overlap by 1-3-nm head-to-tail, whereas laterally they align antiparallel both unstaggered and approximately half-staggered.     

The role of the C9b domain in the binding of C9 molecules to EAC1-8 defined by monoclonal antibodies to C9

Three mAb to human C9, X195, X197, and P40 were used to analyze the roles of the C9a and C9b domains in the reaction of the C9 molecule with sensitized sheep E bearing C1 to C8 (EAC1-8). X195 bound to NH2-terminal (C9a) fragments, and X197 bound to COOH-terminal (C9b) fragments obtained by cleavage of C9 with alpha-thrombin or trypsin. P40 recognized the epitope on the C9b fragment obtained by alpha-thrombin cleavage but did not react with the NH2-terminal or COOH-terminal fragment obtained by trypsin cleavage. In this respect, P40 differed from mAb to C9 reported previously. P40 almost completely inhibited the hemolytic activity of C9. X195 and X197 also inhibited C9 activity, but less effectively than P40. C9 molecules bound to P40 could not bind to EAC1-8 cells. C9 bound to X197 could not bind rapidly to EAC1-8, but prolonged incubation of the C9-X197 complex with EAC1-8 caused considerable lysis of the cells. C9 molecules bound to X195 could bind rapidly to EAC1-8, but their lytic activity was partially inhibited by the bound antibody. From these results, it is concluded that the C9b but not C9a domain contributes to the binding of C9 to EAC1-8 and that the epitope recognized by P40 or a closely adjacent site may be the binding site of C9 molecule to EAC1-8.     

Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs. Critical role of NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities.

Interleukin-8 (IL-8) is an inflammatory mediator that stimulates neutrophil migration and functional activation. Analogs of human IL-8 were chemically synthesized, purified, and compared with the full-length 72-residue synthetic IL-8 for their ability to stimulate neutrophil chemotaxis and exocytosis as measured by assaying for release of elastase, as well as their binding to specific receptors in competition assays. Analogs corresponding to the less abundant natural forms, 3-72, 4-72, and 77-residue IL-8, were evaluated and the 3-72 and 4-72 had 2-5-fold higher potencies, whereas the 77-residue IL-8 was 2-fold less potent. A major finding was that NH2-terminal residues 4, 5, and 6 were absolutely essential for IL-8 activity and receptor binding. Quantitative dissociation of elastase release and chemotaxis activity was detected with 5-72, which compared with 1-72, was 80-fold less potent in the elastase assay, but was only slightly less potent in stimulating chemotaxis. IL-8 6-72 lacked all the biological activities tested but had detectable receptor binding activity. The NH2-terminal peptide, AVLPRSAKEL, lacked activity and receptor binding, suggesting that the NH2-terminal region alone is not sufficient for function. Comparison of analogs shortened at the COOH terminus showed that potency was progressively reduced as the COOH-terminal residues were excluded. However activity was retained in an analog (1-51) with the entire COOH-terminal alpha helix and beta turn missing. A peptide corresponding to the COOH-terminal 22 residues, although inactive alone, synergized with the 1-51 analog in stimulating elastase release. The results suggest that the NH2-terminal residues 4, 5, and 6, which are disordered in the IL-8 solution structure, are directly involved in receptor binding, but the COOH-terminal alpha helix is probably important for stabilizing the three-dimensional structure. Other regions within residues 7-51 are also functionally important.     

Regulation of ATPase and chaperone cycle of DnaK from Thermus thermophilus by the nucleotide exchange factor GrpE

The nucleotide binding and release cycle of the molecular chaperone DnaK is regulated by the accessory proteins GrpE and DnaJ, also called co-chaperones. The concerted action of the nucleotide exchange factor GrpE and the ATPase-stimulating factor DnaJ determines the ratio of the two nucleotide states of DnaK, which differ in their mode of interaction with unfolded proteins. In the Escherichia coli system, the stimulation by these two antagonists is comparable in magnitude, resulting in a balance of the two nucleotide states of DnaK(Eco) in the absence and the presence of co-chaperones.The regulation of the DnaK chaperone system from Thermus thermophilus is apparently substantially different. Here, DnaJ does not stimulate the DnaK-mediated ATP hydrolysis and thus does not appear to act as an antagonist of the nucleotide exchange factor GrpE(Tth). This raises the question of whether T. thermophilus GrpE stimulates nucleotide exchange to a smaller degree as compared to the E. coli system and how the corresponding rates relate to intrinsic ATPase and ATP binding as well as luciferase refolding kinetics of T. thermophilus DnaK.We determined dissociation constants as well as kinetic constants that describe the interactions between the T. thermophilus molecular chaperone DnaK, its nucleotide exchange factor GrpE and the fluorescent ADP analogue N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine-5'-diphosphate by isothermal equilibrium titration calorimetry and stopped-flow kinetic experiments and investigated the influence of T. thermophilus DnaJ on the DnaK nucleotide cycle.The interaction of GrpE with the DnaK.ADP complex versus nucleotide-free DnaK can be described by a simple equilibrium system, where GrpE reduces the affinity of DnaK for ADP by a factor of about 10. Kinetic experiments indicate that the maximal acceleration of nucleotide release by GrpE is 80,000-fold at a saturating GrpE concentration.Our experiments show that in T. thermophilus, although the thermophilic DnaK system displays no stimulation of the DnaK-ATPase activity by DnaJ, nucleotide exchange is still efficiently stimulated by GrpE. This indicates that two counteracting factors are not absolutely necessary to maintain a functional and regulated chaperone cycle. This conclusion is corroborated by data that show that the slower ATPase cycle of the DnaK system as well as of heterologous T. thermophilus DnaK/E. coli DnaK systems is directly reflected in altered refolding kinetics of firefly luciferase but not necessarily in refolding yields.     

Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation

DnaK, the prokaryotic Hsp70 molecular chaperone, requires the nucleotide exchange factor and heat shock protein GrpE to release ADP. GrpE and DnaK are tightly associated molecules with an extensive protein-protein interface, and in the absence of ADP, the dissociation constant for GrpE and DnaK is in the low nanomolar range. GrpE reduces the affinity of DnaK for ADP, and the reciprocal linkage is also true: ADP reduces the affinity of DnaK for GrpE. The energetic contributions of GrpE side-chains to GrpE-DnaK binding were probed by alanine-scanning mutagenesis. Sedimentation velocity (SV) analytical ultracentrifugation (AUC) was used to measure the equilibrium constants (Keq) for GrpE binding to the ATPase domain of DnaK in the presence of ADP. ADP-bound DnaK is the natural target of GrpE, and the addition of ADP (final concentration of 5 microM) to the preformed GrpE-DnaK(ATPase) complexes allowed the equilibrium association constants to be brought into an experimentally accessible range. Under these experimental conditions, the substitution of one single GrpE amino acid residue, arginine 183 with alanine, resulted in a GrpE-DnaK(ATPase) complex that was weakly associated (Keq =9.4 x 10(4) M). This residue has been previously shown to be part of a thermodynamic linkage between two structural domains of GrpE: the thermosensing long helices and the C-terminal beta-domains. Several other GrpE side-chains were found to have a significant change in the free energy of binding (DeltaDeltaG approximately 1.5 to 1.7 kcal mol(-1)), compared to wild-type GrpE.DnaK(ATPase) in the same experimental conditions. Overall, the strong interactions between GrpE and DnaK appear to be dominated by electrostatics, not unlike barnase and barstar, another well-characterized protein-protein interaction. GrpE, an inherent thermosensor, exhibits non-Arrhenius behavior with respect to its nucleotide exchange function at bacterial heat shock temperatures, and mutation of several solvent-exposed side-chains located along the thermosensing indicated that these residues are indeed important for GrpE-DnaK interactions.     

Phosphatidylinositol glycan (PI-G) anchored membrane proteins. Amino acid requirements adjacent to the site of cleavage and PI-G attachment in the COOH-terminal signal peptide.

Secreted proteins are processed from a nascent form that contains an NH2-terminal signal peptide. During processing, the latter is cleaved by a specific NH2-terminal signal peptidase. The nascent form of phosphatidylinositol glycan (PI-G) tailed proteins contain both an NH2- and a COOH-terminal signal peptide. The two signal peptides have much in common, such as size and hydrophobicity. The COOH-terminal peptide is also cleaved during processing. We propose that the amino acid in a nascent protein that ultimately combines with the PI-G moiety be designated the omega site. Amino acids adjacent and COOH-terminal to the omega site would then be omega + 1, omega + 2, etc. In previous studies, we showed that allowable substitutions at the omega site of an engineered form of placental alkaline phosphatase (miniPLAP) are limited to 6 small amino acids. In the present study, mutations were made at the omega + 1 and omega + 2 sites. At the omega + 1 site, processing to varying degrees was observed with 8 of the 9 amino acids substituted for alanine, the normal constituent. Only the proline mutant showed no processing. By contrast, the only substituents permitted at the omega + 2 site were glycine and alanine, with only trace activity observed with serine and cysteine. Thus, just as there is a -1, -3 rule for predicting cleavage by NH2-terminal signal peptidase, there appears to be a comparable omega, omega + 2 rule for predicting cleavage/PI-G addition by COOH-terminal signal transamidase.     

Localization of the actin-binding sites of Acanthamoeba myosin IB and effect of limited proteolysis on its actin-activated Mg2+-ATPase activity

Acanthamoeba myosin IB contains a 125-kDa heavy chain that has high actin-activated Mg2+-ATPase activity when 1 serine residue is phosphorylated. The heavy chain contains two F-actin-binding sites, one associated with the catalytic site and a second which allows myosin IB to cross-link actin filaments but has no direct effect on catalytic activity. Tryptic digestion of the heavy chain initially produces an NH2-terminal 62-kDa peptide that contains the ATP-binding site and the regulatory phosphorylation site, and a COOH-terminal 68-kDa peptide. F-actin, in the absence of ATP, protects this site and tryptic cleavage then produces an NH2-terminal 80-kDa peptide. Both the 62- and the 80-kDa peptides retain the (NH+4,EDTA)-ATPase activity of native myosin IB and both bind to F-actin in an ATP-sensitive manner. However, only the 80-kDa peptide retains a major portion of the actin-activated Mg2+-ATPase activity. This activity requires phosphorylation of the 80-kDa peptide by myosin I heavy chain kinase but, in contrast to the activity of intact myosin IB, it has a simple, hyperbolic dependence on the concentration of F-actin. Also unlike myosin IB, the 80-kDa peptide cannot cross-link F-actin filaments indicating the presence of only a single actin-binding site. These results allow the assignment of the actin-binding site involved in catalytic activity to the region near, and possibly on both sides of, the tryptic cleavage site 62 kDa from the NH2 terminus, and the second actin-binding site to the COOH-terminal 45-kDa domain. Thus, the NH2-terminal 80 kDa of the myosin IB heavy chain is functionally similar to the 93-kDa subfragment 1 of muscle myosin and most likely has a similar organization of functional domains.     

Mammalian heterogeneous nuclear ribonucleoprotein A1. Nucleic acid binding properties of the COOH-terminal domain

A1 is a core protein of the eukaryotic heterogeneous nuclear ribonucleoprotein complex and is under study here as a prototype single-stranded nucleic acid-binding protein. A1 is a two-domain protein, NH2-terminal and COOH-terminal, with highly conserved primary structure among vertebrate homologues sequenced to date. It is well documented that the NH2-terminal domain has single-stranded DNA and RNA binding activity. We prepared a proteolytic fragment of rat A1 representing the COOH-terminal one-third of the intact protein, the region previously termed COOH-terminal domain. This purified fragment of 133 amino acids binds to DNA and also binds tightly to the fluorescent reporter poly(ethenoadenylate), which is used to access binding parameters. In solution with 0.41 M NaCl, the equilibrium constant is similar to that observed with A1 itself, and binding is cooperative. The purified COOH-terminal fragment can be photochemically cross-linked to bound nucleic acid, confirming that COOH-terminal fragment residues are in close contact with the polynucleotide lattice. These binding results with isolated COOH-terminal fragment indicate that the COOH-terminal domain in intact A1 can contribute directly to binding properties. Contact between both COOH-terminal domain and NH2-terminal domain residues in an intact A1:poly(8-azidoadenylate) complex was confirmed by photochemical cross-linking.     

The COOH-terminal domain of the JIL-1 histone H3S10 kinase interacts with histone H3 and is required for correct targeting to chromatin

The JIL-1 histone H3S10 kinase in Drosophila localizes specifically to euchromatic interband regions of polytene chromosomes and is enriched 2-fold on the male X chromosome. JIL-1 can be divided into four main domains including an NH(2)-terminal domain, two separate kinase domains, and a COOH-terminal domain. Our results demonstrate that the COOH-terminal domain of JIL-1 is necessary and sufficient for correct chromosome targeting to autosomes but that both COOH- and NH(2)-terminal sequences are necessary for enrichment on the male X chromosome. We furthermore show that a small 53-amino acid region within the COOH-terminal domain can interact with the tail region of histone H3, suggesting that this interaction is necessary for the correct chromatin targeting of the JIL-1 kinase. Interestingly, our data indicate that the COOH-terminal domain alone is sufficient to rescue JIL-1 null mutant polytene chromosome defects including those of the male X chromosome. Nonetheless, we also found that a truncated JIL-1 protein which was without the COOH-terminal domain but retained histone H3S10 kinase activity was able to rescue autosome as well as partially rescue male X polytene chromosome morphology. Taken together these findings indicate that JIL-1 may participate in regulating chromatin structure by multiple and partially redundant mechanisms.