Modular Domain Organization Research Articles

Structure and functions of His domain protein tyrosine phosphatase in receptor trafficking and cancer 1.

Cell surface receptors trigger the activation of signaling pathways to regulate key cellular processes, including cell survival and proliferation. Internalization, sorting, and trafficking of activated receptors, therefore, play a major role in the regulation and attenuation of cell signaling. Efficient sorting of endocytosed receptors is performed by the ESCRT machinery, which targets receptors for degradation by the sequential establishment of protein complexes. These events are tightly regulated and malfunction of ESCRT components can lead to abnormal trafficking and sustained signaling and promote tumor formation or progression. In this review, we analyze the modular domain organization of the alternative ESCRT protein HD-PTP and its role in receptor trafficking and tumorigenesis.

Differential thermodynamic behaviours of the extra-cellular regions of two Ser/Thr PrkC kinases revealed by calorimetric studies

Eukaryotic-type Ser/Thr protein-kinases are critical mediators of developmental changes and host pathogen interactions in bacteria. Although with lower abundance compared to their homologues from eukaryotes, Ser/Thr protein-kinases (STPK) are widespread in gram positive bacteria, where they regulate several cellular functions. STPKs belong to the protein kinase family named as one-component signal transduction systems, which combine both sensing and regulating properties. Thermodynamic investigations of sensing extra-cellular portions of two important Ser-Thr kinases, PrkC, from Staphylococcus aureus and Bacillus subtilis were conducted by differential scanning calorimetry (DSC) and circular dichroism (CD) melting measurements, coupled with modelling studies. The study of thermodynamic properties of the two domains is challenging since they share a modular domain organization. Consistently, DSC and CD data show that they present similar thermodynamic behaviours and that folding/unfolding transitions do not fit a two-state folding model. However, the thermal unfolding of the two proteins is differentially sensitive to pH. In particular, their unfolding is characteristic of modular structures at the neutral pH, with independent contributions of individual domains to folding. Differently, a cooperative unfolding is evidenced at acidic pH for the B. subtilis member, suggesting that a significant interaction between domains becomes valuable.

Two-state dynamics of the SH3-SH2 tandem of Abl kinase and the allosteric role of the N-cap.

The regulation and localization of signaling enzymes is often mediated by accessory modular domains, which frequently function in tandems. The ability of these tandems to adopt multiple conformations is as important for proper regulation as the individual domain specificity. A paradigmatic example is Abl, a ubiquitous tyrosine kinase of significant pharmacological interest. SH3 and SH2 domains inhibit Abl by assembling onto the catalytic domain, allosterically clamping it in an inactive state. We investigate the dynamics of this SH3-SH2 tandem, using microsecond all-atom simulations and differential scanning calorimetry. Our results indicate that the Abl tandem is a two-state switch, alternating between the conformation observed in the structure of the autoinhibited enzyme and another configuration that is consistent with existing scattering data for an activated form. Intriguingly, we find that the latter is the most probable when the tandem is disengaged from the catalytic domain. Nevertheless, an amino acid stretch preceding the SH3 domain, the so-called N-cap, reshapes the free-energy landscape of the tandem and favors the interaction of this domain with the SH2-kinase linker, an intermediate step necessary for assembly of the autoinhibited complex. This allosteric effect arises from interactions between N-cap and the SH2 domain and SH3-SH2 connector, which involve a phosphorylation site. We also show that the SH3-SH2 connector plays a determinant role in the assembly equilibrium of Abl, because mutations thereof hinder the engagement of the SH2-kinase linker. These results provide a thermodynamic rationale for the involvement of N-cap and SH3-SH2 connector in Abl regulation and expand our understanding of the principles of modular domain organization.

N-Terminal Disordered Domain of Saccharomyces cerevisiae Hop1 Protein Is Dispensable for DNA Binding, Bridging, and Synapsis of Double-Stranded DNA Molecules But Is Necessary for Spore Formation

The cytological architecture of the synaptonemal complex (SC), a meiosis-specific proteinaceous structure, is evolutionarily conserved among eukaryotes. However, little is known about the biochemical properties of SC components or the mechanisms underlying their roles in meiotic chromosome synapsis and recombination. Functional analysis of Saccharomyces cerevisiae Hop1, a key structural component of SC, has begun to reveal important insights into its function in interhomolog recombination. Previously, we showed that Hop1 is a structure-specific DNA-binding protein, exhibits higher binding affinity for the Holliday junction, and induces structural distortion at the core of the junction. Furthermore, Hop1 promotes DNA condensation and intra- and intermolecular synapsis between duplex DNA molecules. Here, we show that Hop1 possesses a modular domain organization, consisting of an intrinsically disordered N-terminal domain and a protease-resistant C-terminal domain (Hop1CTD). Furthermore, we found that Hop1CTD exhibits strong homotypic as well as heterotypic protein-protein interactions, and its biochemical activities were similar to those of the full-length Hop1 protein. However, Hop1CTD failed to complement the meiotic recombination defects of the Δhop1 strain, indicating that both N- and C-terminal domains of Hop1 are essential for meiosis and spore formation. Altogether, our findings reveal novel insights into the structure-function relationships of Hop1 and help to further our understanding of its role in meiotic chromosome synapsis and recombination.

A dodecameric ring-like structure of the N0 domain of the type II secretin from enterotoxigenic Escherichia coli

In many bacteria, secretins from the type II secretion system (T2SS) function as outer membrane gated channels that enable passage of folded proteins from the periplasm into the extracellular milieu. Cryo-electron microscopy of the T2SS secretin GspD revealed previously the dodecameric cylindrical architecture of secretins, and crystal structures of periplasmic secretin domains showed a modular domain organization. However, no high-resolution experimental data has as yet been provided about how the entire T2SS secretin or its domains are organized in a cylindrical fashion. Here we present a crystal structure of the N0 domain of the T2SS secretin GspD from enterotoxigenic Escherichia coli containing a helix with 12 subunits per turn. The helix has an outer diameter of ∼125Å and a pitch of only 24Å which suggests a model of a cylindrical dodecameric N0 ring whose dimensions correspond with the cryo-electron microscopy map of Vibrio cholerae GspD. The N0 domain is known to interact with the HR domain of the inner membrane T2SS protein GspC. When the new N0 ring model is combined with the known N0·HR crystal structure, a dodecameric double-ring of twelve N0-HR heterodimers is obtained. In contrast, the previously observed compact N0-N1 GspD module is not compatible with the N0 ring. Interestingly, a N0-N1 T3SS homolog is compatible with forming a N0-N1 dodecameric ring, due to a different N0-vs-N1 orientation. This suggests that the dodecameric N0 ring is an important feature of T2SS secretins with periplasmic domains undergoing considerable motions during exoprotein translocation.

The nuclear receptor signalling scaffold: insights from full-length structures

Nuclear receptors (NRs) can be conceptualized as highly dynamic scaffold proteins, where binding of ligand, DNA or transcriptional coregulator proteins can allosterically change the scaffold structure and direct changes in subsequent binding events. In this issue of The EMBO Journal, Orlov et al present the first cryo-EM structure of a NR complex, a technically challenging feat for the 100-kDa complex of the heterodimer of the vitamin D receptor (VDR), Retinoid X receptor (RXR) and their cognate DNA response element. VDR is one of the few NRs for which the hinge between the ligand-binding domain (LBD) and DNA-binding domain is an extended helix, which enforced a bend in VDR/RXR to an L-shaped architecture. The hinge domain is thus a key regulator of the relative orientation of the LBDs to the DNA, which will impact how transcriptional coregulator complexes are oriented towards the chromatin. It further suggests that the positioning of the hinge may serve as a conduit of structural information, determining how specific DNA sequences can modulate activity in the LBDs. Nuclear receptors (NRs) are a superfamily of transcription factors that regulate key aspects of development, metabolism and disease (Huang et al, 2010). NRs typically form transcriptionally active homodimers or heterodimers with another NR, that is, Retinoid X receptor (RXR). The classic modular domain organization of NRs includes a variable N-terminal domain (NTD) that may contain a ligand-independent transcriptional activation function (AF-1); a centrally located zinc-finger DNA-binding domain (DBD); and a C-terminal ligand-binding domain (LBD) that contains ligand-dependent AF-2 (Chandra et al, 2008). The DBD and LBD are well structured and connected via a flexible linker or ‘hinge’ region. NR-interacting proteins include transcriptional coregulators that enzymatically regulate post-translational modification (PTM) of histones and associated proteins (Metivier

The Signaling Adaptor Eps8 Is an Essential Actin Capping Protein for Dendritic Cell Migration

Dendritic cells (DCs) flexibly adapt to different microenvironments by using diverse migration strategies that are ultimately dependent on the dynamics and structural organization of the actin cytoskeleton. Here, we have shown that DCs require the actin capping activity of the signaling adaptor Eps8 to polarize and to form elongated migratory protrusions. DCs from Eps8-deficient mice are impaired in directional and chemotactic migration in 3D invitro and are delayed in reaching the draining lymph node (DLN) invivo after inflammatory challenge. Hence, Eps8-deficient mice are unable to mount a contact hypersensitivity response. We have also shown that the DC migratory defect is cell autonomous and that Eps8 is required for the proper architectural organization of the actin meshwork and dynamics of cell protrusions. Yet, Eps8 is not necessary for antigen uptake, processing, and presentation. Thus, we have identified Eps8 as a unique actin capping protein specifically required for DC migration.

Homology modeling and consensus protein disorder prediction of human filamin.

Filamins are dimeric actin-binding proteins participating in the organization of the actin-based cytoskeleton. Their modular domain organization is made up of an N-terminal actin-binding domain composed of two CH domains followed by flexible rod regions that consist of 24 Ig-like domains. Homology modeling was used to model human filamin using Modeller 9v5. The resulting model assessed by Verify 3D and PROCHECK showed that the final model is reliable. The conformational disorder prediction of human filamin residues were also mapped on the validated structure of human filamin. Prediction of protein disorder in filamin structures will help structural biologists to find suitable targets to be analyzed and for understanding protein function.

Swapping the domains of exoribonucleases RNase II and RNase R: Conferring upon RNase II the ability to degrade ds RNA

RNase II and RNase R are the two E. coli exoribonucleases that belong to the RNase II super family of enzymes. They degrade RNA hydrolytically in the 3' to 5' direction in a processive and sequence independent manner. However, while RNase R is capable of degrading structured RNAs, the RNase II activity is impaired by dsRNAs. The final end-product of these two enzymes is also different, being 4 nt for RNase II and 2 nt for RNase R. RNase II and RNase R share structural properties, including 60% of amino acid sequence similarity and have a similar modular domain organization: two N-terminal cold shock domains (CSD1 and CSD2), one central RNB catalytic domain, and one C-terminal S1 domain. We have constructed hybrid proteins by swapping the domains between RNase II and RNase R to determine which are the responsible for the differences observed between RNase R and RNase II. The results obtained show that the S1 and RNB domains from RNase R in an RNase II context allow the degradation of double-stranded substrates and the appearance of the 2 nt long end-product. Moreover, the degradation of structured RNAs becomes tail-independent when the RNB domain from RNase R is no longer associated with the RNA binding domains (CSD and S1) of the genuine protein. Finally, we show that the RNase R C-terminal Lysine-rich region is involved in the degradation of double-stranded substrates in an RNase II context, probably by unwinding the substrate before it enters into the catalytic cavity.

RNase R mutants elucidate the catalysis of structured RNA: RNA-binding domains select the RNAs targeted for degradation

The RNase II superfamily is a ubiquitous family of exoribonucleases that are essential for RNA metabolism. RNase II and RNase R degrade RNA in the 3'-->5' direction in a processive and sequence-independent manner. However, although RNase R is capable of degrading highly structured RNAs, the RNase II activity is impaired by the presence of secondary structures. RNase II and RNase R share structural properties and have a similar modular domain organization. The eukaryotic RNase II homologue, Rrp44/Dis3, is the catalytic subunit of the exosome, one of the most important protein complexes involved in the maintenance of the correct levels of cellular RNAs. In the present study, we constructed truncated RNase II and RNase R proteins and point mutants and characterized them regarding their exoribonucleolytic activity and RNA-binding ability. We report that Asp280 is crucial for RNase R activity without affecting RNA binding. When Tyr324 was changed to alanine, the final product changed from 2 to 5 nt in length, showing that this residue is responsible for setting the end-product. We have shown that the RNB domain of RNase II has catalytic activity. The most striking result is that the RNase R RNB domain itself degrades double-stranded substrates even in the absence of a 3'-overhang. Moreover, we have demonstrated for the first time that the substrate recognition of RNase R depends on the RNA-binding domains that target the degradation of RNAs that are 'tagged' by a 3'-tail. These results can have important implications for the study of poly(A)-dependent RNA degradation mechanisms.

Allosteric and binding properties of Asp1–Glu382 truncated recombinant human serum albumin – an optical and NMR spectroscopic investigation

Human serum albumin (HSA) is known for its exceptional ligand-binding capacity; indeed, its modular domain organization provides a variety of ligand-binding sites. Its flexible modular structure involves more than the immediate vicinity of the binding site(s), affecting the ligand-binding properties of the whole protein. Here, biochemical characterization by (1)H-NMR relaxometry and optical spectroscopy of a truncated form of HSA (tHSA) encompassing domains I and II (Asp1-Glu382) is reported. Removal of the C-terminal domain III results in a number of contacts that involve domain I (containing the heme site) and domain II (containing the warfarin site) being lost; however, the allosteric linkage between heme and warfarin sites is maintained. tHSA shows a nuclear magnetic relaxation dispersion profile similar to that of HSA, and displays increased affinity for ibuprofen, warfarin, and heme, suggesting that the fold is preserved. Moreover, the allosteric properties that make HSA a peculiar monomeric protein and account for the regulation of ligand-binding modes by heterotropic interactions are maintained after removal of domain III. Therefore, tHSA is a valuable model with which to investigate allosteric properties of HSA, allowing independent analysis of the linkages between different drug-binding sites.

The N1317H Substitution Associated with Leber Congenital Amaurosis Results in Impaired Interdomain Packing in Human CRB1 Epidermal Growth Factor-like (EGF) Domains

The calcium-binding epidermal growth factor-like (cbEGF) domain is a widely occurring module in proteins of diverse function. Amino acid substitutions that disrupt its structure or calcium affinity have been associated with various disorders. The extracellular portion of CRB1, the human homologue of Drosophila Crumbs, exhibits a modular domain organization that includes EGF and cbEGF domains. The N1317H substitution in the 19th cbEGF domain of CRB1 is associated with the serious visual disorder Leber congenital amaurosis. We have investigated the structure and Ca(2+) binding of recombinant wild-type and N1317H CRB1 fragments (EGF18-cbEGF19) using NMR and find that Ca(2+) binding is altered, resulting in disruption of long range interactions between adjacent EGF domains in CRB1. From these observations, we propose that this substitution affects the structural integrity of CRB1 in the inter-photoreceptor matrix of the retina, where it is expressed. Furthermore, we identify disease-causing substitutions in other cbEGF-containing proteins that are likely to result in similar disruption of interdomain packing, supporting the hypothesis that the tandem cbEGF domain linkages are critical for the structure and function of proteins containing cbEGF domains.

Restriction of human immunodeficiency virus type 1 by TRIM-CypA occurs with rapid kinetics and independently of cytoplasmic bodies, ubiquitin, and proteasome activity.

TRIM-CypA is an owl monkey-specific variant of the retrovirus restriction factor TRIM5alpha. Here, we exploit its modular domain organization and cyclosporine sensitivity to probe the kinetics and mechanism of TRIM5-mediated restriction. Time of addition/withdrawal experiments reveal that inhibition of incoming human immunodeficiency virus type 1 capsids by TRIM-CypA occurs within minutes of their delivery to the target cell cytoplasm. However, while TRIM-CypA restriction is partly dependent on a RING domain, restriction occurs independently of the ubiquitin/proteasome system. Moreover, tagged TRIM-CypA proteins can be fully active as restriction factors without forming cytoplasmic bodies.

Hrp59, an hnRNP M protein in Chironomus and Drosophila, binds to exonic splicing enhancers and is required for expression of a subset of mRNAs

Here, we study an insect hnRNP M protein, referred to as Hrp59. Hrp59 is relatively abundant, has a modular domain organization containing three RNA-binding domains, is dynamically recruited to transcribed genes, and binds to premRNA cotranscriptionally. Using the Balbiani ring system of Chironomus, we show that Hrp59 accompanies the mRNA from the gene to the nuclear envelope, and is released from the mRNA at the nuclear pore. The association of Hrp59 with transcribed genes is not proportional to the amount of synthesized RNA, and in vivo Hrp59 binds preferentially to a subset of mRNAs, including its own mRNA. By coimmunoprecipitation of Hrp59–RNA complexes and microarray hybridization against Drosophila whole-genome arrays, we identify the preferred mRNA targets of Hrp59 in vivo and show that Hrp59 is required for the expression of these target mRNAs. We also show that Hrp59 binds preferentially to exonic splicing enhancers and our results provide new insights into the role of hnRNP M in splicing regulation.

Letter to the Editor: Resonance assignments of the double-stranded RNA-binding of adenosine deaminase acting on RNA 2 (ADAR2)

Adenosine deaminases that act on RNA (ADARs) convert adenosine to inosine (A-to-I) by hydrolytic deamination in cellular and viral RNA transcripts containing either perfect or imperfect RNA duplexes (Bass, 2002). A-to-I editing can be either specific or non-specific, deaminating up to 50% of the adenosine residues within a perfect RNA duplex, while modifying a single or limited set of adenosine residues within imperfect double-stranded RNA (dsRNA) regions containing bulges, loops, and mismatches (Bass, 2002). The majority of non-selective editing occurs in untranslated regions (UTRs) and introns where large regular duplexes are formed. Such modifications can modulate gene silencing triggered by intramolecular structures in mRNA (Tonkin and Bass, 2003), the nuclear retention of hyperedited RNA transcripts, or participate in the antiviral response by extensive modification of viral RNAs. Selective editing has been shown to take place largely within codons, so that multiple RNA and protein isoforms can be created from a single genomic locus. For example, ADARs have been shown to produce functionally important isoforms for numerous proteins involved in synaptic neurotransmission, including ligand and voltage-gated ion channels and G-protein coupled receptors (Bass, 2002). Like many RNA-binding proteins, ADARs display a modular domain organization, having two or three tandem copies of double-stranded RNA-binding domain (dsRBDs) at its N-terminal, and a C-terminal adenosine deaminase domain. The dsRBDs of ADARs play an important role in modulating the editing selectivity of ADARs (Carlson et al., 2003). To gain insight into this intriguing protein-RNA recognition process, we have initiated an NMR study of the two dsRBDs of rat ADAR2 (74–301) (designated dsRBD12). Here, we report H, C, and N resonance assignments of the dsRBD12 of ADAR2.

Distinct Sequence Motifs within the 68-kDa Subunit of Cleavage Factor Im Mediate RNA Binding, Protein-Protein Interactions, and Subcellular Localization

Cleavage factor I(m) (CF I(m)) is required for the first step in pre-mRNA 3'-end processing and can be reconstituted in vitro from its heterologously expressed 25- and 68-kDa subunits. The binding of CF I(m) to the pre-mRNA is one of the earliest steps in the assembly of the cleavage and polyadenylation machinery and facilitates the recruitment of other processing factors. We identified regions in the subunits of CF I(m) involved in RNA binding, protein-protein interactions, and subcellular localization. CF I(m)68 has a modular domain organization consisting of an N-terminal RNA recognition motif and a C-terminal alternating charge domain. However, the RNA recognition motif of CF I(m)68 on its own is not sufficient to bind RNA but is necessary for association with the 25-kDa subunit. RNA binding appears to require a CF I(m)68/25 heterodimer. Whereas multiple protein interactions with other 3'-end-processing factors are detected with CF I(m)25, CF I(m)68 interacts with SRp20, 9G8, and hTra2beta, members of the SR family of splicing factors, via its C-terminal alternating charge domain. This domain is also required for targeting CF I(m)68 to the nucleus. However, CF I(m)68 does not concentrate in splicing speckles but in foci that partially colocalize with paraspeckles, a subnuclear component in which other proteins involved in transcriptional control and RNA processing have been found.

Inter- and intragenus structural variations in caliciviruses and their functional implications.

The family Caliciviridae is divided into four genera and consists of single-stranded RNA viruses with hosts ranging from humans to a wide variety of animals. Human caliciviruses are the major cause of outbreaks of acute nonbacterial gastroenteritis, whereas animal caliciviruses cause various host-dependent illnesses with a documented potential for zoonoses. To investigate inter- and intragenus structural variations and to provide a better understanding of the structural basis of host specificity and strain diversity, we performed structural studies of the recombinant capsid of Grimsby virus, the recombinant capsid of Parkville virus, and San Miguel sea lion virus serotype 4 (SMSV4), which are representative of the genera Norovirus (genogroup 2), Sapovirus, and Vesivirus, respectively. A comparative analysis of these structures was performed with that of the recombinant capsid of Norwalk virus, a prototype member of Norovirus genogroup 1. Although these capsids share a common architectural framework of 90 dimers of the capsid protein arranged on a T=3 icosahedral lattice with a modular domain organization of the subunit consisting of a shell (S) domain and a protrusion (P) domain, they exhibit distinct differences. The distally located P2 subdomain of P shows the most prominent differences both in shape and in size, in accordance with the observed sequence variability. Another major difference is in the relative orientation between the S and P domains, particularly between those of noroviruses and other caliciviruses. Despite being a human pathogen, the Parkville virus capsid shows more structural similarity to SMSV4, an animal calicivirus, suggesting a closer relationship between sapoviruses and animal caliciviruses. These comparative structural studies of caliciviruses provide a functional rationale for the unique modular domain organization of the capsid protein with an embedded flexibility reminiscent of an antibody structure. The highly conserved S domain functions to provide an icosahedral scaffold; the hypervariable P2 subdomain may function as a replaceable module to confer host specificity and strain diversity; and the P1 subdomain, located between S and P2, provides additional fine-tuning to position the P2 subdomain.

Domain-specific Interactions of Human HP1-type Chromodomain Proteins and Inner Nuclear Membrane Protein LBR

HP1-type chromodomain proteins self-associate as well as interact with the inner nuclear membrane protein LBR (lamin B receptor) and transcriptional coactivators TIF1alpha and TIF1beta. The domains of these proteins that mediate their various interactions have not been entirely defined. HP1-type proteins are predicted by hydrophobic cluster analysis to consist of two homologous but distinct globular domains, corresponding to the chromodomain and chromo shadow domain, separated by a hinge region. We show here that the chromo shadow domain mediates the self-associations of HP1-type proteins and is also necessary for binding to LBR both in vitro and in the yeast two-hybrid assay. Hydrophobic cluster analysis also predicts that the nucleoplasmic amino-terminal portion of LBR contains two globular domains separated by a hinge region. The interactions of the LBR domains with an HP1-type protein were also analyzed by the yeast two-hybrid and in vitro binding assays, which showed that a portion of the second globular domain is necessary for binding. The modular domain organization of HP1-type proteins and LBR can explain some of the diverse protein-protein interactions at the chromatin-lamina-membrane interface of the nuclear envelope.

Analysis of the architecture of the transcription factor sigma N (sigma 54) and its domains by circular dichroism.

Enhancer-dependent transcription in bacteria requires the alternative transcription factor sigma N (sigma 54), which forms an RNA polymerase holoenzyme that binds promoters as a transcriptionally inactive complex. We have examined the structure of sigma N by circular dichroism (CD) analysis. The sigma N protein and its domains are well structured in the absence of the core RNA polymerase subunits or promoter DNA. Denaturation of sigma N by temperature as followed by changes in CD shows a concomitant loss of secondary and tertiary structures with a melting temperature of 36 degrees C. The secondary structure displays a two-state melting curve with a second Tm of 85 degrees C. The amino-terminal Region I activation domain together with the acidic Region II does not contribute to the two-state melting. In marked contrast, the integrity of the C-terminal DNA-binding domain is required for the two-state melting. Measurements of pKb also demonstrated that a C-terminal part of sigma N, but not regions I or I + II, is required for the structural integrity of sigma N at high pH. Measurements of pKa suggested that alpha-helical structures are important in sigma N for the establishment of tertiary structural elements. The tertiary structure near ultraviolet CD signals of sigma N do not require regions I or I + II but were strongly diminished by C-terminal truncation of sigma N. Promoter DNA binding resulted in a conformational change in sigma N, permitting the determination of a binding constant. A typical B-DNA conformation was adopted by the promoter DNA. Implications for the modular domain organization of sigma N, the function of C-terminal sequences, and domain communication and its role in activation of transcription are discussed.