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.     

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.     

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.     

AH/PH domain-mediated interaction between Akt molecules and its potential role in Akt regulation.

The cytoplasmic serine-threonine protein kinase coded for by the c-akt proto-oncogene features a protein kinase C-like catalytic domain and a unique NH2-terminal domain (AH domain). The AH domain is a member of a domain superfamily whose prototype was observed in pleckstrin (pleckstrin homology, or PH, domain). In this communication, we present evidence that the AH/PH domain is a domain of protein-protein interaction which mediates the formation of Akt protein complexes. The interaction between c-akt AH/PH domains is highly specific, as determined by the failure of this domain to bind AKT2. The AH/PH domain-mediated interactions depend on the integrity of the entire domain. Akt molecules with deletions of the NH2-terminal portion (amino acids 11 to 60) and AH/PH constructs with deletions of the C-terminal portion of this domain (amino acids 107 to 147) fail to interact with c-akt. To determine the significance of these findings, we carried out in vitro kinase assays using Akt immunoprecipitates from serum-starved and serum-starved, platelet-derived growth factor-stimulated NIH 3T3 cells. Addition of maltose-binding protein-AH/PH fusion recombinant protein, which is expected to bind Akt, to the immunoprecipitates from serum-starved cells induced the activation of the Akt kinase.     

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.     

Activation of the cAMP-specific phosphodiesterase PDE4D3 by phosphorylation. Identification and function of an inhibitory domain.

Splicing variants of type 4 phosphodiesterases (PDE4) are regulated by phosphorylation. In these proteins, a conserved region is located between the amino-terminal domain, which is the target for phosphorylation, and the catalytic domain. Previous studies have indicated that nested deletions encompassing this region cause an increase in catalytic activity, suggesting this domain exerts an inhibitory constraint on catalysis. Here, we have further investigated the presence and function of this domain. A time-dependent increase in hydrolytic activity was observed when PDE4D3 from FRTL-5 cells was incubated with the endoproteinase Lys-C. The activation was abolished by protease inhibitors and was absent when a phosphorylated enzyme was used. Western blot analysis with PDE4D-specific antibodies indicated the Lys-C treatment separates the catalytic domain of PDE4D3 from the inhibitory domain. Incubation with antibodies recognizing an epitope within this domain caused a 3- to 4-fold increase in activity of native or recombinant PDE4D3. Again, PDE activation by these antibodies had properties similar to, and not additive with, the activation by protein kinase A phosphorylation. An interaction between the inhibitory domain and both regulatory and catalytic domains of PDE4D3 was detected by the yeast two-hybrid system. Mutations of Ser54 to Ala in the regulatory domain decreased or abolished this interaction, whereas mutations of Ser54 to the negatively charged Asp strengthened it. These data strongly support the hypothesis that an inhibitory domain is present in PDE4D and that phosphorylation of the regulatory domain causes activation of the enzyme by modulating the interaction between inhibitory and catalytic domains.     

Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design

The DVD-Ig^TM protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.     

Resuscitation-promoting factors possess a lysozyme-like domain

The novel bacterial cytokine family--resuscitation-promoting factors (Rpfs)--share a conserved domain of uncharacterized function. Predicting the structure of this domain suggests that Rpfs possess a lysozyme-like domain. The model highlights the good conservation of residues involved in catalysis and substrate binding. A lysozyme-like function makes sense for this domain in the light of experimental characterization of the biological function of Rpfs.     

The Capping Domain in RalF Regulates Effector Functions

The Legionella pneumophila effector protein RalF functions as a guanine nucleotide exchange factor (GEF) that activates the host small GTPase protein ADP-ribosylation factor (Arf), and recruits this host protein to the vacuoles in which this pathogen resides. GEF activity is conferred by the Sec7 domain located in the N-terminal region of RalF. Structural studies indicate that the C-terminal region of RalF makes contacts with residues in the Sec7 domain important for Arf interactions. Theoretically, the C-terminal region of RalF could prevent nucleotide exchange activity by blocking the ability of Arf to interact with the Sec7 domain. For this reason, the C-terminal region of RalF has been termed a capping domain. Here, the role of the RalF capping domain was investigated by comparing biochemical and effector activities mediated by this domain in both the Legionella RalF protein (LpRalF) and in a RalF ortholog isolated from the unrelated intracellular pathogen Rickettsia prowazekii (RpRalF). These data indicate that both RalF proteins contain a functional Sec7 domain and that the capping domain regulates RalF GEF activity. The capping domain has intrinsic determinants that mediate localization of the RalF protein inside of host cells and confer distinct effector activities. Localization mediated by the capping domain of LpRalF enables the GEF to modulate membrane transport in the secretory pathway, whereas, the capping domain of RpRalF enables this bacterial GEF to modulate actin dynamics occurring near the plasma membrane. Thus, these data reveal that divergence in the function of the C-terminal capping domain alters the in vivo functions of the RalF proteins.     

Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design

The DVD-Ig™ protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.Key words: dual variable domain immunoglobulin, DVD-Ig, immunotherapy, variable domain, antibody engineering, dual-specific, linker     

On the interaction of the finger and the kringle-2 domain of tissue-type plasminogen activator with fibrin. Inhibition of kringle-2 binding to fibrin by epsilon-amino caproic acid

The binding of tissue-type plasminogen activator (t-PA) to fibrin is mediated both by its finger domain and by its kringle-2 domain. In this report, we investigate the relative affinities of these domains for lysine. Human recombinant t-PA deletion-mutant proteins were prepared and their ability to bind to lysine-Sepharose was investigated. Mutants containing the kringle-2 domain bound to lysine-Sepharose, whereas mutants lacking this domain but containing the finger domain, the epidermal growth factor domain or the kringle-1 domain did not bind to lysine-Sepharose. Mutant proteins containing the kringle-2 domain could be specifically eluted from lysine-Sepharose with epsilon-amino caproic acid. This lysine derivative also abolished fibrin binding by the kringle-2 domain but had no effect on the fibrin-binding property of the finger domain. Thus, a lysine-binding site is involved in the interaction of the kringle-2 domain with fibrin but not in the interaction of the finger domain with fibrin. The implications of the nature of these two distinct interactions of t-PA with fibrin on plasminogen activation by t-PA will be discussed.     

Crystal structure of the bovine mitochondrial elongation factor Tu.Ts complex

The three-dimensional structure of the bovine mitochondrial elongation factor (EF)-Tu.Ts complex (EF-Tumt.Tsmt) has been determined to 2.2-A resolution using the multi-wavelength anomalous dispersion experimental method. This complex provides the first insight into the structure of EF-Tsmt. EF-Tsmt is similar to Escherichia coli and Thermus thermophilus EF-Ts in the amino-terminal domain. However, the structure of EF-Tsmt deviates considerably in the core domain with a five-stranded beta-sheet forming a portion of subdomain N of the core. In E. coli EF-Ts, this region is composed of a three-stranded sheet. The coiled-coil domain of the E. coli EF-Ts is largely eroded in EF-Tsmt, in which it consists of a large loop packed against subdomain C of the core. The conformation of bovine EF-Tumt in complex with EF-Tsmt is distinct from its conformation in the EF-Tumt.GDP complex. When domain III of bovine EF-Tumt.GDP is superimposed on domain III of EF-Tumt in the EF-Tumt.Tsmt complex, helix B from domain I is also almost superimposed. However, the rest of domain I is rotated relative to this helix toward domain II, which itself is rotated toward domain I relative to domain III. Extensive contacts are observed between the amino-terminal domain of EF-Tsmt and domain I of EF-Tumt. Furthermore, the conserved TDFV sequence of EF-Tsmt also contacts domain I with the side chain of Asp139 contacting helix B of EF-Tumt and inserting the side chain of Phe140 between helices B and C. The structure of the EF-Tumt.Tsmt complex provides new insights into the nucleotide exchange mechanism and provides a framework for explaining much of the mutational data obtained for this complex.     

Alternative splicing of the first F3 domain from chicken collagen XIV affects cell adhesion and heparin binding

The N terminus of chicken collagen XIV is subject to alternative splicing. The longer isoform contains a fibronectin type III (F3) domain at its N terminus, whereas the shorter isoform is lacking this domain. Alternative splicing of the F3 domain is developmentally regulated. At early embryonic stages, both isoforms are expressed, whereas after hatching only the longer isoform is expressed. When immobilized on plastic dishes, the recombinant F3 domain promotes the adhesion of mesenchymal cells. Attachment to this domain is specifically inhibited by heparin but not by other glycosaminoglycans. Molecular modeling studies illustrate that the first F3 domain harbors a positively charged groove, which may accommodate the negatively charged heparin chain. Site-directed mutagenesis of a single lysine residue within this groove abolishes the cell binding activity but does not affect the heparin binding activity. Cell binding and heparin binding are therefore two functionally distinct properties shared by the N-terminal F3 domain. When full-length collagen XIV polypeptides that either contain or lack the first F3 domain are tested on heparin-Sepharose, a pronounced difference in their relative affinity is observed. Thus, alternative splicing of the N-terminal F3 domain influences the interaction of this FACIT (fibril-associated collagens with interrupted triple helices) collagen with cells and with glycosaminoglycans.     

SSD: sterol-sensing direct

Cholesterol homeostasis is established by a complex of three proteins, one of which contains a hydrophobic domain previously termed a sterol-sensing domain. New biochemical studies of this domain demonstrate direct high-affinity binding of the sterol-sensing domain to sterol.     

Four-helix bundle: a ubiquitous sensory module in prokaryotic signal transduction

MOTIVATION: Transmembrane chemoreceptors in Escherichia coli utilize ligand-binding domains for detecting various external signals. The structure of this domain in the E.coli aspartate receptor, Tar, is known and its signal transduction mechanism is under investigation. Current domain models for this important sensory module are inaccurate and, therefore, cannot reveal the distribution of this domain within the current genomic landscape. RESULTS: We carried out sensitive and exhaustive PSI-BLAST searches initiated with the sequence corresponding to a known structure of the four-helix, ligand-binding domain of the aspartate chemoreceptor. From the resulting sequences, we built a multiple sequence alignment for this domain family, which confirmed that the current TarH model is erroneous and fails to detect most of the domain homologs. In the process, we developed a technique that visualizes the secondary structure prediction of each protein sequence in order to improve the multiple sequence alignment. We found that the four-helix up-and-down bundle represents a large domain family and includes representatives of all major classes of prokaryotic signal transduction, namely histidine kinases, di-guanylate cyclases and chemotaxis receptors.