Roles of functional and structural domains of hepatocyte growth factor activator inhibitor type 1 in the inhibition of matriptase

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound, Kunitz-type serine protease inhibitor. HAI-1 inhibits serine proteases that have potent pro-hepatocyte growth factor-converting activity, such as the membrane-type serine protease, matriptase. HAI-1 comprises an N-terminal domain, followed by an internal domain, first protease inhibitory domain (Kunitz domain I), low-density lipoprotein receptor A module (LDLRA) domain, and a second Kunitz domain (Kunitz domain II) in the extracellular region. Our aim was to assess the roles of these domains in the inhibition of matriptase. Soluble forms of recombinant rat HAI-1 mutants made up with various combinations of domains were produced, and their inhibitory activities toward the hydrolysis of a chromogenic substrate were analyzed using a soluble recombinant rat matriptase. Kunitz domain I exhibited inhibitory activity against matriptase, but Kunitz domain II did not. The N-terminal domain and Kunitz domain II decreased the association rate between Kunitz domain I and matriptase, whereas the internal domain increased this rate. The LDLRA domain suppressed the dissociation of the Kunitz domain I-matriptase complex. Surprisingly, an HAI-1 mutant lacking the N-terminal domain and Kunitz domain II showed an inhibitor constant of 1.6 pm, and the inhibitory activity was 400 times higher in this HAI-1 mutant than in the mutant with all domains. These findings, together with the known occurrence of an HAI-1 species lacking the N-terminal domain and Kunitz domain II in vivo, suggest that the domain structure of HAI-1 is organized in a way that allows HAI-1 to flexibly control matriptase activity.     

Stable interdomain interaction within the cytoplasmic domain of CD45 increases enzyme stability

CD45 is a leukocyte-specific, two domain transmembrane tyrosine phosphatase. Co-purification of a recombinant protein containing the first phosphatase domain of CD45 (6His-D1) with a recombinant protein containing the second phosphatase domain (GST-D2) from E. coli indicated a stable interaction which resulted in increased stability of the active phosphatase domain present in 6His-D1. This interaction was not dependent on the acidic region unique to CD45 domain 2, but was affected by a destabilizing point mutation (Q1180G) in GST-D2. CD45 domain 2 enhanced phosphatase activity of the first domain in the full length cytoplasmic domain protein, whereas a chimeric protein with the SH2 domain of p56(lck) in place of the CD45 C-terminal region did not. Thus the C-terminal domain of CD45 associates with the N-terminal domain and this stabilizes the active phosphatase domain. A single destabilizing point mutation in the second domain is sufficient to attenuate this effect.     

Functional characterization of the CFTR R domain using CFTR/MDR1 hybrid and deletion constructs.

To improve our insight into the structure and function of the CFTR R domain, deletion and hybrid constructs in which different parts of the R domain were deleted or replaced by the MDR1 linker domain, and vice versa, were made. Replacement of the linker domain by the R domain did not result in a decrease and replacement of the CFTR R domain by the linker domain did not result in an increase of maturation efficiency, when compared to the respective wild-type proteins. This indicates that the R domain is not responsible for the high degree of degradation observed for CFTR translation products in the ER, but rather the overall structure or sequences located outside the R domain. Replacing the C-terminal part of the R domain (amino acids 780-830) by the MDR1 linker domain resulted in the appearance of PKA-dependent whole cell chloride currents which were not significantly different from wild-type CFTR currents. This might indicate that the PKA sites present in the linker domain are functional and that not the exact sequence of the C-terminal part of the R domain is important, but rather the presence of PKA sites and the length. Moreover, when this hybrid construct was PKC-stimulated, chloride currents were activated. Although these PKC-induced currents were lower than the PKA-induced ones, this again indicates that the linker domain is functional in this hybrid construct. Taken together, these results suggest that the MDR1 linker domain can substitute for part of the regulatory domain of the CFTR protein.     

Role of the pleckstrin homology domain in intersectin-L Dbl homology domain activation of Cdc42 and signaling

Intersectin-long (ITSN-L) contains the invariant Dbl homology (DH) and pleckstrin homology (PH) domain structure characteristic of the majority of Dbl family proteins. This strict domain topography suggests that the PH domain serves an essential, conserved function in the regulation of the intrinsic guanine nucleotide exchange activity of the DH domain. We evaluated the role of the PH domain in regulating the DH domain function of ITSN-L. Surprisingly, we found that the PH domain was dispensable for guanine nucleotide exchange activity on Cdc42 in vitro, yet the PH domain enhanced the ability of the DH domain to activate Cdc42 signaling in vivo. PH domains can interact with phosphoinositide substrates and products of phosphatidylinositol 3-kinase (PI3K). However, PI3K activation did not modulate ITSN-L DH domain function in vivo.     

Regulation of Raf through phosphorylation and N terminus-C terminus interaction

Raf kinase is a key component in regulating the MAPK pathway. B-Raf has been reported as an oncogene and is mutated in 60% of human melanomas. The main focus of Raf regulation studies has been on phosphorylation, dephosphorylation, and scaffolding proteins; however, Raf also has its own auto-regulatory domain. Removal of the N-terminal regulatory domain, initially discovered in the viral Raf oncogene (v-Raf), results in a kinase domain with high basal activity independent of Ras activation. In this report, we show that activating phosphorylations are still required for activity of the truncated C-terminal kinase domain (called 22W). The interaction between the N-terminal regulatory domain and the C-terminal kinase domain is disrupted by activated Ras. Mutations in the Ras binding domain, cysteine-rich domain, or S259A do not affect the inhibition of 22W by the N-terminal domain. When phosphomimetic residues are substituted at the activating sites (DDED) in 22W, this results in a higher basal activity that is no longer inhibited by expression of the N-terminal domain, although binding to the N-terminal domain still occurs. Although the interaction between 22W/DDED and the N-terminal domain may be in a different conformation, the interaction is still disrupted by activated Ras. These data demonstrate that N-terminal domain binding to the kinase domain inhibits the activity of the kinase domain. However, this inhibition is relieved when the C-terminal kinase domain is activated by phosphorylation.     

Definition of the p53 functional domains necessary for inducing apoptosis

The p53 protein contains several functional domains necessary for inducing cell cycle arrest and apoptosis. The C-terminal basic domain within residues 364-393 and the proline-rich domain within residues 64-91 are required for apoptotic activity. In addition, activation domain 2 within residues 43-63 is necessary for apoptotic activity when the N-terminal activation domain 1 within residues 1-42 is deleted (DeltaAD1) or mutated (AD1(-)). Here we have discovered that an activation domain 2 mutation at residues 53-54 (AD2(-)) abrogates the apoptotic activity but has no significant effect on cell cycle arrest. We have also found that p53-(DeltaAD2), which lacks activation domain 2, is inert in inducing apoptosis. p53-(AD2(-)DeltaBD), which is defective in activation domain 2 and lacks the C-terminal basic domain, p53-(DeltaAD2DeltaBD), which lacks both activation domain 2 and the C-terminal basic domain, and p53-(DeltaPRDDeltaBD), which lacks both the proline-rich domain and the C-terminal basic domain, are also inert in inducing apoptosis. All four mutants are still capable of inducing cell cycle arrest, albeit to a lesser extent than wild-type p53. Interestingly, we have found that deletion of the N-terminal activation domain 1 alleviates the requirement of the C-terminal basic domain for apoptotic activity. Thus, we have generated a small but potent p53-(DeltaAD1DeltaBD) molecule. Furthermore, we have determined that at least two of the three domains (activation domain 1, activation domain 2, and the proline-rich domain), are required for inducing cell cycle arrest. Taken together, our results suggest that activation domain 2 and the proline-rich domain form an activation domain for inducing pro-apoptotic genes or inhibiting anti-apoptotic genes. The C-terminal basic domain is required for maintaining this activation domain competent for transactivation or transrepression.     

Solution and membrane-bound chaperone activity of the diphtheria toxin translocation domain towards the catalytic domain

During cell intoxication by diphtheria toxin, endosome acidification triggers the translocation of the catalytic (C) domain into the cytoplasm. This event is mediated by the translocation (T) domain of the toxin. Previous work suggested that the T domain acts as a chaperone for the C domain during membrane penetration of the toxin. Using partitioning experiments with lipid vesicles, fluorescence spectroscopy, and a lipid vesicle leakage assay, we characterized the dominant behavior of the T domain over the C domain during the successive steps by which these domains interact with a membrane upon acidification: partial unfolding in solution and during membrane binding, and then structural rearrangement during penetration into the membrane. To this end, we compared, for each domain, isolated or linked together in a CT protein (the toxin lacking the receptor-binding domain), each of these steps. The behavior of the T domain is marginally modified by the presence or absence of the C domain, whereas that of the C domain is greatly affected by the presence of the T domain . All of the steps leading to membrane penetration of the C domain are triggered at higher pH by the T domain , by 0.5-1.6 pH units. The T domain stabilizes the partially folded states of the C domain corresponding to each step of the process. The results unambiguously demonstrate that the T domain acts as a specialized pH-dependent chaperone for the C domain. Interestingly, this chaperone activity acts on very different states of the protein: in solution, membrane-bound, and membrane-inserted.     

Design, synthesis, structure and binding properties of PDZ binding, cyclic β-finger peptides

Protein interaction domains (PIDs) play a critical role in signal transduction. One PID of great interest is the PDZ domain, a 100 amino-acid-residue domain. Most PDZ domains recognize short, C-terminal peptide motives. In the heterodimer of the nNOS-PDZ domain and the α-syntrophin-PDZ domain, however, one PDZ domain forms a β-finger that binds to the other PDZ domain. We show here that cyclic peptides derived from the β-finger of the nNOS-PDZ domain can bind the syntrophin-PDZ domain in the same manner as the whole domain. The structure of three “finger-peptides” of different size has been determined and the binding investigated using calorimetry and NMR-titration experiments.     

The cooperative role of OsCnfU-1A domain I and domain II in the iron sulphur cluster transfer process as revealed by NMR

OsCnfU-1A is a chloroplast-type Nfu-like protein that consists of tandem repeats sharing high sequence homology. Domain I of this protein, but not domain II, has a C-X-X-C motif that is thought to assemble an iron-sulphur cluster. Herein we report the solution structure of OsCnfU-1A domain I (73-153). Although OsCnfU-1A domain I is structurally similar to OsCnfU-1A domain II (154-226), the electrostatic surface potential of the 2 domains differs. Domain I has an acidic surface, whereas that of domain II is predominantly basic. Chemical shift perturbation studies on OsCnfU-1A domain I and domain II with ferredoxin revealed negligible chemical shift changes in domain I, whereas much larger chemical shift changes were observed in domain II. The residues with larger chemical shift changes were located on the basic surface of domain II. Considering that ferredoxin is predominantly negatively charged, we propose the following hypothesis: First, an iron-sulphur cluster is assembled on domain I. Next, domain II interacts with the ferredoxin, thus tethering domain I close to the ferredoxin. Finally, domain I transfers the iron-sulphur cluster to the ferredoxin. Thus, domain II facilitates the efficient transfer of the iron-sulphur cluster from domain I to the ferredoxin.     

Influence of Ser/Pro-rich domain and kinase domain of double cortin-like protein kinase on microtubule-binding activity

Doublecortin-like protein kinase (DCLK) is a Ser/Thr protein kinase predominantly expressed in brain. DCLK is composed of three functional domains; the N-terminal doublecortin-like (DC) domain, the C-terminal kinase domain and Ser/Pro-rich (SP) domain in between DC and kinase domains. Although the DC domain is known to mediate microtubule association, functional roles of the SP domain and the kinase domain on microtubule association is not known. In this study, we investigated the microtubule-binding activity of zebrafish DCLK (zDCLK) using various deletion mutants and chimeric proteins. The microtubule-binding activity of various mutants of zDCLK was assessed both by immunocytochemical analysis and by biochemical analysis using detergent extraction method. When the kinase domain was removed from zDCLK, the microtubule-binding activity was significantly enhanced. Although the zDCLK(DC + SP) mutant showed a strong microtubule-binding activity, the DC domain alone showed much lower microtubule-binding activity, indicating that the SP domain of zDCLK plays a role in enhancing microtubule-binding activity of the DC domain. These results suggest that both the kinase domain and the SP domain are involved in regulating the microtubule-binding activity of DCLK.     

Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein

SlyD, the sensitive-to-lysis protein from Escherichia coli, consists of two domains. They are not arranged successively along the protein chain, but one domain, the “insert-in-flap” (IF) domain, is inserted internally as a guest into a surface loop of the host domain, which is a prolyl isomerase of the FK506 binding protein (FKBP) type. We used SlyD as a model to elucidate how such a domain insertion affects the stability and folding mechanism of the host and the guest domain. For these studies, the two-domain protein was compared with a single-domain variant SlyDΔIF, SlyD* without the chaperone domain (residues 1-69 and 130-165) in which the IF domain was removed and replaced by a short loop, as present in human FKBP12. Equilibrium unfolding and folding kinetics followed an apparent two-state mechanism in the absence and in the presence of the IF domain. The inserted domain decreased, however, the stability of the host domain in the transition region and decelerated its refolding reaction by about 10-fold. This originates from the interruption of the chain connectivity by the IF domain and its inherent instability. To monitor folding processes in this domain selectively, a Trp residue was introduced as fluorescent probe. Kinetic double-mixing experiments revealed that, in intact SlyD, the IF domain folds and unfolds about 1000-fold more rapidly than the FKBP domain, and that it is strongly stabilized when linked with the folded FKBP domain. The unfolding limbs of the kinetic chevrons of SlyD show a strong downward curvature. This deviation from linearity is not caused by a transition-state movement, as often assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentrations. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded.     

Crystal structures of pyruvate phosphate dikinase from maize revealed an alternative conformation in the swiveling-domain motion

Pyruvate phosphate dikinase (PPDK) reversibly catalyzes the conversion of ATP, phosphate, and pyruvate into AMP, pyrophosphate, and phosphoenolpyruvate (PEP), respectively. Since the nucleotide binding site (in the N-terminal domain) and the pyruvate/PEP binding site (in the C-terminal domain) are separated by approximately 45 A, it has been proposed that an intermediary domain, called the central domain, swivels between these remote domains to transfer the phosphate. However, no direct structural evidence for the swiveling central domain has been found. In this study, the crystal structures of maize PPDK with and without PEP have been determined at 2.3 A resolution. These structures revealed that the central domain is located near the pyruvate/PEP binding C-terminal domain, in contrast to the PPDK from Clostridium symbiosum, wherein the central domain is located near the nucleotide-binding N-terminal domain. Structural comparisons between the maize and C. symbiosum PPDKs demonstrated that the swiveling motion of the central domain consists of a rotation of at least 92 degrees and a translation of 0.5 A. By comparing the maize PPDK structures with and without PEP, we have elucidated the mode of binding of PEP to the C-terminal domain and the induced conformational changes in the central domain.     

Residues in a conserved α-helical segment are required for cleavage but not secretion of an Escherichia coli serine protease autotransporter passenger domain

Autotransporters are a superfamily of virulence factors produced by Gram-negative bacteria that are comprised of an N-terminal extracellular domain (passenger domain) and a C-terminal β barrel domain (β domain) that resides in the outer membrane (OM). The β domain promotes the translocation of the passenger domain across the OM by an unknown mechanism. Available evidence indicates that an α-helical segment that spans the passenger domain-β domain junction is embedded inside the β domain at an early stage of assembly. Following its secretion, the passenger domain of the serine protease autotransporters of the Enterobacteriaceae (SPATEs) and the pertactin family of Bordetella pertussis autotransporters is released from the β domain through an intrabarrel autoproteolytic cleavage of the α-helical segment. Although the mutation of conserved residues that surround the cleavage site has been reported to impair both the translocation and cleavage of the passenger domain of a SPATE called Tsh, we show here that the mutation of the same residues in another SPATE (EspP) affects only passenger domain cleavage. Our results strongly suggest that the conserved residues are required to position the α-helical segment for the cleavage reaction and are not required to promote passenger domain secretion.     

Solution structure of the GUCT domain from human RNA helicase II/Gu beta reveals the RRM fold, but implausible RNA interactions

Human RNA helicase II/Gu alpha (RH-II/Gu alpha) and RNA helicase II/Gu beta (RH-II/Gu beta) are paralogues that share the same domain structure, consisting of the DEAD box helicase domain (DEAD), the helicase conserved C-terminal domain (helicase_C), and the GUCT domain. The N-terminal regions of the RH-II/Gu proteins, including the DEAD domain and the helicase_C domain, unwind double-stranded RNAs. The C-terminal tail of RH-II/Gu alpha, which follows the GUCT domain, folds a single RNA strand, while that of RH-II/Gu beta does not, and the GUCT domain is not essential for either the RNA helicase or foldase activity. Thus, little is known about the GUCT domain. In this study, we have determined the solution structure of the RH-II/Gu beta GUCT domain. Structural calculations using NOE-based distance restraints and residual dipolar coupling-based angular restraints yielded a well-defined structure with beta-alpha-alpha-beta-beta-alpha-beta topology in the region for K585-A659, while the Pfam HMM algorithm defined the GUCT domain as G571-E666. This structure-based domain boundary revealed false positives in the sequence homologue search using the HMM definition. A structural homology search revealed that the GUCT domain has the RRM fold, which is typically found in RNA-interacting proteins. However, it lacks the surface-exposed aromatic residues and basic residues on the beta-sheet that are important for the RNA recognition in the canonical RRM domains. In addition, the overall surface of the GUCT domain is fairly acidic, and thus the GUCT domain is unlikely to interact with RNA molecules. Instead, it may interact with proteins via its hydrophobic surface around the surface-exposed tryptophan.     

Comparative analysis of SET domain proteins in maize and Arabidopsis reveals multiple duplications preceding the divergence of monocots and dicots

Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.     

The RAC binding domain/IRSp53-MIM homology domain of IRSp53 induces RAC-dependent membrane deformation

The concave surface of the crescent-shaped Bin-amphiphysin-Rvs (BAR) domain is postulated to bind to the cell membrane to induce membrane deformation of a specific curvature. The Rac binding (RCB) domain/IRSp53-MIM homology domain (IMD) has a dimeric structure that is similar to the structure of the BAR domain; however, the RCB domain/IMD has a "zeppelin-shaped" dimer. Interestingly, the RCB domain/IMD of IRSp53 possesses Rac binding, membrane binding, and actin filament binding abilities. Here we report that the RCB domain/IMD of IRSp53 induces membrane deformation independent of the actin filaments in a Rac-dependent manner. In contrast to the BAR domain, the RCB domain/IMD did not cause long tubulation of the artificial liposomes; however, the Rac binding domain caused the formation of small buds on the liposomal surface. When expressed in cells, the Rac binding domain induced outward protrusion of the plasma membrane in a direction opposite to that induced by the BAR domain. Mapping of the amino acids responsible for membrane deformation suggests that the convex surface of the Rac binding domain binds to the membrane in a Rac-dependent manner, which may explain the mechanism of the membrane deformation induced by the RCB domain/IMD.     

Hierarchy of membrane-targeting signals of phospholipase D1 involving lipid modification of a pleckstrin homology domain

The amino terminus of phospholipase D1 (PLD1) contains three potential membrane-interacting determinants: a phox homology (PX) domain, a pleckstrin homology (PH) domain and two adjacent cysteines at positions 240 and 241 within the PH domain that are fatty acylated in vivo. To understand how these determinants contribute to membrane localization, we have mutagenized critical residues of the PLD1 PH domain in the wild type or palmitate-free background in the intact protein, in a fragment that deletes the first 210 amino acids including the PX domain, and in the isolated PH domain. Mutants were expressed in COS-7 cells and examined for membrane residence, intracellular localization, palmitoylation, and catalytic activity. Our results are as follows. 1) Mutagenesis of critical residues of the PH domain results in redistribution of PLD1 from membranes to cytosol, independently of fatty acylation sites. Importantly, PH domain mutants in the wild type background showed greatly reduced fatty acylation, despite the presence of all relevant cysteines. 2) The isolated PH domain did not co-localize with PLD1 and was not palmitoylated. 3) The PX deletion mutant showed similar distribution and palmitoylation to the intact protein. Interestingly, PH domain mutants in this background showed significant palmitoylation and incomplete cytosolic redistribution. 4) PH domain mutants in the wild type or palmitate-free background maintained catalytic activity. We propose that membrane targeting of PLD1 involves a hierarchy of signals with a functional PH domain allowing fatty acylation leading to strong membrane binding. The PX domain may modulate function of the PH domain.     

孢子体型自交不亲和反应臂重复蛋白ARC1

臂重复蛋白ARC1（arm repeat containing 1,ARC1）是孢子体型自交不亲和信号传导途径下游非常重要的蛋白质因子,由臂重复结构域、U-box结构域、亮氨酸拉链,卷曲螺旋结构域、1个核定位信号和2个核输出信号组成,其中臂重复结构域和U—box结构域起主要功能。ARC1具有E3泛素连接酶活性,能够促进自交不亲和反应（self-incompatibility,SI）中的信号传导元件泛素化并降解。本文综述了ARC1蛋白的结构和功能,旨在阐明它在SI反应中的作用。