Small-Angle X-Ray Scattering Reveals Compact Domain-Domain Interactions in the N-Terminal Region of Filamin C

Filamins are multi-domain, actin cross-linking, and scaffolding proteins. In addition to the actin cross-linking function, filamins have a role in mechanosensor signaling. The mechanosensor function is mediated by domain-domain interaction in the C-terminal region of filamins. Recently, we have shown that there is a three-domain interaction module in the N-terminal region of filamins, where the neighboring domains stabilize the structure of the middle domain and thereby regulate its interaction with ligands. In this study, we have used small-angle X-ray scattering as a tool to screen for potential domain-domain interactions in the N-terminal region. We found evidence of four domain-domain interactions with varying flexibility. These results confirm our previous study showing that domains 3, 4, and 5 exist as a compact three domain module. In addition, we report interactions between domains 11–12 and 14–15, which are thus new candidate sites for mechanical regulation.     

Differential mechanical stability of filamin A rod segments

Prompted by recent reports suggesting that interaction of filamin A (FLNa) with its binding partners is regulated by mechanical force, we examined mechanical properties of FLNa domains using magnetic tweezers. FLNa, an actin cross-linking protein, consists of two subunits that dimerize through a C-terminal self-association domain. Each subunit contains an N-terminal spectrin-related actin-binding domain followed by 24 immunoglobulinlike (Ig) repeats. The Ig repeats in the rod 1 segment (repeats 1–15) are arranged as a linear array, whereas rod 2 (repeats 16–23) is more compact due to interdomain interactions. In the rod 1 segment, repeats 9–15 augment F-actin binding to a much greater extent than do repeats 1–8. Here, we report that the three segments are unfolded at different forces under the same loading rate. Remarkably, we found that repeats 16–23 are susceptible to forces of ∼10 pN or even less, whereas the repeats in the rod 1 segment can withstand significantly higher forces. The differential force response of FLNa Ig domains has broad implications, since these domains not only support the tension of actin network but also interact with many transmembrane and signaling proteins, mostly in the rod 2 segment. In particular, our finding of unfolding of repeats 16–23 at ∼10 pN or less is consistent with the hypothesized force-sensing function of the rod 2 segment in FLNa.     

1H, 13C and 15N resonance assignments of the human filamin A tandem immunoglobulin-like domains 16–17 and 18–19

Filamins are large actin-binding and cross-linking proteins which act as linkers between the cytoskeleton and various signaling proteins. Filamin A (FLNa) is the most abundant of the three filamin isoforms found in humans. FLNa contains an N-terminal actin-binding domain and 24 immunoglobulin-like (Ig) domains. The Ig domains are responsible for the FLNa dimerization and most of the interactions that FLNa has with numerous other proteins. There are several crystal and solution structures from isolated single Ig domains of filamins in the PDB database, but only few from longer constructs. Here, we present nearly complete chemical shift assignments of FLNa tandem Ig domains 16–17 and 18–19. Chemical shift mapping between FLNa tandem Ig domain 16–17 and isolated domain 17 suggests a novel domain–domain interaction mode.     

Molecular structure of the rod domain of dictyostelium filamin

Dictyostelium discoideum filamin (ddFLN) is a two-chain F-actin crosslinking protein with an N-terminal actin-binding domain and a rod domain constructed from six tandem repeats of a 100 residue motif that has an immunoglobulin (Ig) fold. We report the 2.8 A resolution crystal structure of a homodimer of rod repeats 4, 5 and 6. The two chains are arranged in an antiparallel fashion and form an elongated element, which is shortened, however, compared to a fully extended, linear configuration because the long axis of each Ig domain is arranged at an angle to the long axis of the rod. Same arrangement of repeats should also be present in the rod domain of human FLNa, much longer than Dictyostelium FLN, which forms an extended structure able to crosslink F-actin chains over distances of more than 1000 A.     

Structural basis for dimerization of the Dictyostelium gelation factor (ABP120) rod.

Gelation factor (ABP120) is one of the principal actin-cross-linking proteins of Dictyostelium discoideum. The extended molecule has an N-terminal 250-residue actin-binding domain and a rod constructed from six 100-residue repeats that have an Ig fold. The ability to dimerize is crucial to the actin cross-linking function of gelation factor and is mediated by the rod in which the two chains are arranged in an antiparallel fashion. We report the 2.2 A resolution crystal structure of rod domains 5 and 6, which shows that dimerization is mediated primarily by rod domain 6 and is the result of a double edge-to-edge extension of beta-sheets. Thus, contrary to earlier proposals, the chains of the dimeric gelation factor molecule overlap only within domain 6, and domains 1-5 do not pair with domains from the other chain. This information allows construction of a model of the gelation factor molecule and suggests how the chains in the related molecule filamin (ABP280) may interact.     

Monomeric and homotrimeric solution structures of truncated human peroxidasin 1 variants

Human peroxidasin 1 is a multidomain peroxidase situated in the basement membrane. The iron enzyme with covalently bound heme oxidizes bromide to hypobromous acid which facilitates the formation of distinct sulfilimine cross-links in the collagen IV network and therefore contributes to its mechanical stability. Additional to the catalytically active peroxidase domain peroxidasin comprises a leucine rich repeat domain, four Ig domains and a C-terminal von Willebrand factor type C module (VWC). Peroxidasin has been shown to form homotrimers involving two redox-sensitive cysteine residues and to undergo posttranslational C-terminal proteolytic cleavage. The present study on several recombinantly produced truncated peroxidasin variants showed that the VWC is not required for trimer formation whereas the alpha-helical linker region located between the peroxidase domain and the VWC is crucial for trimerization. Our data furthermore implies that peroxidasin oligomerization occurs intracellularly before C-terminal cleavage. For the first time we present overall solution structures of monomeric and trimeric truncated peroxidasin variants which were determined by rotary shadowing combined with transmission electron microscopy and by small-angle X-ray scattering (SAXS). A triangular arrangement of the peroxidase domains to each other within the homotrimer was revealed and this structure was confirmed by a model of trimeric peroxidase domains. Our SAXS data showed that the Ig domains are highly flexible and interact with the peroxidase domain and that within the homotrimer each alpha-helical linker region interacts with the respective adjacent peroxidase domain. The implications of our findings on the structure-function relationship of peroxidasin are discussed.     

Association between the first two immunoglobulin-like domains of the neural cell adhesion molecule N-CAM.

The extracellular domain of N-CAM contains five immunoglobulin-like (Ig) and two fibronectin type III-like domains and facilitates cell-cell binding through multiple, weak interdomain interactions. NMR spectroscopy indicated that the two N-terminal Ig-like domains from chicken N-CAM (Ig I and Ig II) interact with millimolar affinity. Physico-chemical studies show that this interaction is significantly amplified when the domains are covalently linked, consistent with an antiparallel domain arrangement. The binding of the two individual domains and the dimerization of the concatenated protein were essentially independent of salt, up to a concentration of 200 mM. The residues in Ig I involved in the interaction map to the BED strands of the beta sandwich, and delineate a largely hydrophobic patch.     

The structure of Prp40 FF1 domain and its interaction with the crn-TPR1 motif of Clf1 gives a new insight into the binding mode of FF domains

The yeast splicing factor Prp40 (pre-mRNA processing protein 40) consists of a pair of WW domains followed by several FF domains. The region comprising the FF domains has been shown to associate with the 5' end of U1 small nuclear RNA and to interact directly with two proteins, the Clf1 (Crooked neck-like factor 1) and the phosphorylated repeats of the C-terminal domain of RNA polymerase II (CTD-RNAPII). In this work we reported the solution structure of the first FF domain of Prp40 and the identification of a novel ligand-binding site in FF domains. By using chemical shift assays, we found a binding site for the N-terminal crooked neck tetratricopeptide repeat of Clf1 that is distinct and structurally separate from the previously identified CTD-RNAPII binding pocket of the FBP11 (formin-binding protein 11) FF1 domain. No interaction, however, was observed between the Prp40 FF1 domain and three different peptides derived from the CTD-RNAPII protein. Indeed, the equivalent CTD-RNAPII-binding site in the Prp40 FF1 domain is predominantly negatively charged and thus unfavorable for an interaction with phosphorylated peptide sequences. Sequence alignments and phylogenetic tree reconstructions using the FF domains of three functionally related proteins, Prp40, FBP11, and CA150, revealed that Prp40 and FBP11 are not orthologous proteins and supported the different ligand specificities shown by their respective FF1 domains. Our results also revealed that not all FF domains in Prp40 are functionally equivalent. We proposed that at least two different interaction surfaces exist in FF domains that have evolved to recognize distinct binding motifs.     

Neural cell adhesion molecule (N-CAM) homophilic binding mediated by the two N-terminal Ig domains is influenced by intramolecular domain-domain interactions

The mechanism by which the neural cell adhesion molecule, N-CAM, mediates homophilic interactions between cells has been variously attributed to an isologous interaction of the third immunoglobulin (Ig) domain, to reciprocal binding of the two N-terminal Ig domains, or to reciprocal interactions of all five Ig domains. Here, we have used a panel of recombinant proteins in a bead binding assay, as well as transfected and primary cells, to clarify the molecular mechanism of N-CAM homophilic binding. The entire extracellular region of N-CAM mediated bead aggregation in a concentration- and temperature-dependent manner. Interactions of the N-terminal Ig domains, Ig1 and Ig2, were essential for bead binding, based on deletion and mutation experiments and on antibody inhibition studies. These findings were largely in accord with aggregation experiments using transfected L cells or primary chick brain cells. Additionally, maximal binding was dependent on the integrity of the intramolecular domain-domain interactions throughout the extracellular region. We propose that these interactions maintain the relative orientation of each domain in an optimal configuration for binding. Our results suggest that the role of Ig3 in homophilic binding is largely structural. Several Ig3-specific reagents failed to affect N-CAM binding on beads or on cells, while an inhibitory effect of an Ig3-specific monoclonal antibody is probably due to perturbations at the Ig2-Ig3 boundary. Thus, it appears that reciprocal interactions between Ig1 and Ig2 are necessary and sufficient for N-CAM homophilic binding, but that maximal binding requires the quaternary structure of the extracellular region defined by intramolecular domain-domain interactions.     

Acan125 binding to the SH3 domain of acanthamoeba myosin-IC

The domain organization of Acanthamoeba myosin-I, an oligomodular motor protein, includes a potentially important protein interaction module that is mostly uncharacterized. The Src homology 3, SH3, domain of myosin-I binds Acan125, a protein containing at least two consensus ligand binding domains: C-terminal SH3 binding motifs (PXXP) and N-terminal leucine-rich repeats. We report the first affinities determined for an SH3 domain of any myosin, namely, K(d) = 7 microM for a 21-residue synthetic peptide based on the PXXP domain sequence and K(d) = 0.15 microM for the PXXP domain included in the C-terminus of Acan125. These values are consistent with affinities reported for peptides and proteins that associate with SH3. By deletional analysis we show that only the PXXP domain is required for Acan125 to interact with the SH3 domain of Acanthamoeba myosin-IC (AmyoC(SH3)). The synthetic peptide described above at a concentration near the K(d) for SH3 binding blocked the interaction between native AmyoC and Acan125, mapping the interaction to the PXXP domain of Acan125 and the SH3 domain of myosin-I. These results are consistent with prototypical SH3 binding and suggest that a PXXP module is both necessary and sufficient to interact with an SH3 module of myosin-I.     

The MECP2-TRD domain interacts with the DNMT3A-ADD domain at the H3-tail binding site

The DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A-ADD and MECP2-TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull-down, equilibrium peptide binding assays, and structural analyses. The region D529-D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N-terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214–228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3/TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2-TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific binding.     

The spectrin repeat: a structural platform for cytoskeletal protein assemblies

Spectrin repeats are three-helix bundle structures which occur in a large number of diverse proteins, either as single copies or in tandem arrangements of multiple repeats. They can serve structural purposes, by coordination of cytoskeletal interactions with high spatial precision, as well as a 'switchboard' for interactions with multiple proteins with a more regulatory role. We describe the structure of the alpha-actinin spectrin repeats as a prototypical example, their assembly in a defined antiparallel dimer, and the interactions of spectrin repeats with multiple other proteins. The alpha-actinin rod domain shares several features common to other spectrin repeats. (1) The rod domain forms a rigid connection between two actin-binding domains positioned at the two ends of the alpha-actinin dimer. The exact distance and rigidity are important, for example, for organizing the muscle Z-line and maintaining its architecture during muscle contraction. (2) The spectrin repeats of alpha-actinin have evolved to make tight antiparallel homodimer contacts. (3) The spectrin repeats are important interaction sites for multiple structural and signalling proteins. The interactions of spectrin repeats are, however, diverse and defy any simple classification of their preferred interaction sites, which is possible for other domains (e.g. src-homology domains 3 or 2). Nevertheless, the binding properties of the repeats perform important roles in the biology of the proteins where they are found, and lead to the assembly of complex, multiprotein structures involved both in cytoskeletal architecture as well as in forming large signal transduction complexes.     

The inhibitory gamma subunit of the rod cGMP phosphodiesterase binds the catalytic subunits in an extended linear structure

The unique feature of rod photoreceptor cGMP phosphodiesterase (PDE6) is the presence of inhibitory subunits (Pgamma), which interact with the catalytic heterodimer (Palphabeta) to regulate its activity. This uniqueness results in an extremely high sensitivity and sophisticated modulations of rod visual signaling where the Pgamma/Palphabeta interactions play a critical role. The quaternary organization of the alphabetagammagamma heterotetramer is poorly understood and contradictory patterns of interaction have been previously suggested. Here we provide evidence that supports a specific interaction, by systematically and differentially analyzing the Pgamma-binding regions on Palpha and Pbeta through photolabel transfer from various Pgamma positions throughout the entire molecule. The Pgamma N-terminal Val16-Phe30 region was found to interact with the Palphabeta GAFa domain, whereas its C terminus (Phe73-Ile87) interacted with the Palphabeta catalytic domain. The interactions of Pgamma with these two domains were bridged by its central Ser40-Phe50 region through interactions with GAFb and the linker between GAFb and the catalytic domain, indicating a linear and extended interaction between Pgamma and Palphabeta. Furthermore, a photocross-linked product alphabetagamma(gamma) was specifically generated by the double derivatized Pgamma, in which one photoprobe was located in the polycationic region and the other in the C terminus. Taken together the evidence supports the conclusion that each Pgamma molecule binds Palphabeta in an extended linear interaction and may even interact with both Palpha and Pbeta simultaneously.     

The ZASP-like motif in actinin-associated LIM protein is required for interaction with the alpha-actinin rod and for targeting to the muscle Z-line

The Z-line is a specialized structure connecting adjacent sarcomeres in muscle cells. alpha-Actinin cross-links actin filaments in the Z-line. Several PDZ-LIM domain proteins localize to the Z-line and interact with alpha-actinin. Actinin-associated LIM protein (ALP), C-terminal LIM domain protein (CLP36), and Z band alternatively spliced PDZ-containing protein (ZASP) have a conserved region named the ZASP-like motif (ZM) between PDZ and LIM domains. To study the interactions and function of ALP we used purified recombinant proteins in surface plasmon resonance measurements. We show that ALP and alpha-actinin 2 have two interaction sites. The ZM motif was required for the interaction of ALP internal region with the alpha-actinin rod and for targeting of ALP to the Z-line. The PDZ domain of ALP bound to the C terminus of alpha-actinin. This is the first indication that the ZM motif would have a direct role in a protein-protein interaction. These results suggest that the two interaction sites of ALP would stabilize certain conformations of alpha-actinin 2 that would strengthen the Z-line integrity.     

Interactions of the basic N-terminal and the acidic C-terminal domains of the maize chromosomal HMGB1 protein

Maize HMGB1 is a typical member of the family of plant chromosomal HMGB proteins, which have a central high-mobility group (HMG)-box DNA-binding domain that is flanked by a basic N-terminal region and a highly acidic C-terminal domain. The basic N-terminal domain positively influences various DNA interactions of the protein, while the acidic C-terminal domain has the opposite effect. Using DNA-cellulose binding and electrophoretic mobility shift assays, we demonstrate that the N-terminal basic domain binds DNA by itself, consistent with its positive effects on the DNA interactions of HMGB1. To examine whether the negative effect of the acidic C-terminal domain is brought about by interactions with the basic part of HMGB1 (N-terminal region, HMG-box domain), intramolecular cross-linking in combination with formic acid cleavage of the protein was used. These experiments revealed that the acidic C-terminal domain interacts with the basic N-terminal domain. The intramolecular interaction between the two oppositely charged termini of the protein is enhanced when serine residues in the acidic tail of HMGB1 are phosphorylated by protein kinase CK2, which can explain the negative effect of the phosphorylation on certain DNA interactions. In line with that, covalent cross-linking of the two terminal domains resulted in a reduced affinity of HMGB1 for linear DNA. Comparable to the finding with maize HMGB1, the basic N-terminal and the acidic C-terminal domains of the Arabidopsis HMGB1 and HMGB4 proteins interact, indicating that these intramolecular interactions, which can modulate HMGB protein function, generally occur in plant HMGB proteins.     

Structurally unique interaction of RBD-like and PH domains is crucial for yeast pheromone signaling

The Ste5 protein forms a scaffold that associates and regulates the components of the mitogen-activated protein (MAP) kinase cascade that controls mating-pheromone-mediated signaling in the yeast Saccharomyces cerevisiae. Although it is known that the MEK kinase of the pathway, Ste11, associates with Ste5, details of this interaction have not been established. We identified a Ras-binding-domain-like (RBL) region in the Ste11 protein that is required specifically for the kinase to function in the mating pathway. This module is structurally related to domains in other proteins that mediate Ras-MAP kinase kinase kinase associations; however, this RBL module does not interact with Ras, but instead binds the PH domain of the Ste5 scaffold. Structural and functional studies suggest that the key role of this PH domain is to mediate the Ste5–Ste11 interaction. Overall these two evolutionarily conserved modules interact with each other through a unique interface, and thus in the pheromone pathway the structural context of the RBL domain contribution to kinase activation has been shifted through a change of its interaction partner from Ras to a PH domain.     

Filamin isoforms in molluscan smooth muscle

The role of filamin in molluscan catch muscles is unknown. In this work three proteins isolated from the posterior adductor muscle of the sea mussel Mytilus galloprovincialis were identified by MALDI-TOF/TOF MS as homologous to mammalian filamin. They were named FLN-270, FLN-230 and FLN-105, according to their apparent molecular weight determined by SDS-PAGE: 270kDa, 230kDa and 105kDa, respectively. Both FLN-270 and FLN-230 contain the C-terminal dimerization domain and the N-terminal actin-binding domain typical of filamins. These findings, together with the data from peptide mass fingerprints, indicate that FLN-270 and FLN-230 are different isoforms of mussel filamin, with FLN-230 being the predominant isoform in the mussel catch muscle. De novo sequencing data revealed structural differences between both filamin isoforms at the rod 2 segment, the one responsible for the interaction of filamin with the most of its binding partners. FLN270 but not FLN230 was phosphorylated in vitro by cAMP-dependent protein kinase. As for the FLN-105, it would be an N-terminal proteolytic fragment generated from the FLN-270 isoform or a C-terminally truncated variant of filamin. On the other hand, a 45-kDa protein that copurifies with mussel catch muscle filamins was identified as the mussel calponin-like protein. The fact that this protein coelutes with the FLN-270 isoform from a gel filtration chromatography suggests a specific interaction between both proteins.