Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin

Twinfilin is a ubiquitous actin monomer-binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the alpha and beta subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer-sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.     

Regulation of the Cortical Actin Cytoskeleton in Budding Yeast by Twinfilin,a Ubiquitous Actin Monomer-sequestering Protein

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)–twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.     

Conformational dynamics of capping protein and interaction partners: simulation studies

Capping protein (CP) is important for the regulation of actin polymerization. CP binds to the barbed end of the actin filament and prevents actin polymerization. This interaction is modulated through competitive binding by regulatory proteins such as myotrophin (V-1) and the capping protein interacting (CPI) motif from CARMIL. The binding site of myotrophin overlaps with the region of CP that binds to the barbed end of actin filament, whereas CPI binds at a distant site. The binding of CPI to the myotrophin-CP complex dissociates myotrophin from CP. Detailed multicopy molecular dynamics simulations suggest that the binding of CPI shifts the conformational equilibria of CP away from states that favor myotrophin binding. This shift is underpinned by allosteric effects where CPI inhibits CP through suppression of flexibility and disruption of concerted motions that appear to mediate myotrophin binding. Accompanying these effects are changes in electrostatic interactions, notably those involving residue K142β, which appears to play a critical role in regulating flexibility. In addition, accessibility of the site on CP for binding the key hydrophobic residue W8 of myotrophin is modulated by CPI. These results provide insights into the modulation of CP by CPI and myotrophin and indicate the mechanism by which CPI drives the dissociation of the myotrophin-CP complex.     

Advances in Structural Biology and the Application to Biological Filament Systems

Structural biology has experienced several transformative technological advances in recent years. These include: development of extremely bright X‐ray sources (microfocus synchrotron beamlines and free electron lasers) and the use of electrons to extend protein crystallography to ever decreasing crystal sizes; and an increase in the resolution attainable by cryo‐electron microscopy. Here we discuss the use of these techniques in general terms and highlight their application for biological filament systems, an area that is severely underrepresented in atomic resolution structures. We assemble a model of a capped tropomyosin‐actin minifilament to demonstrate the utility of combining structures determined by different techniques. Finally, we survey the methods that attempt to transform high resolution structural biology into more physiological environments, such as the cell. Together these techniques promise a compelling decade for structural biology and, more importantly, they will provide exciting discoveries in understanding the designs and purposes of biological machines.     

Capping Protein Insulates Arp2/3-Assembled Actin Patches from Formins

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Patterns,structures, and amino acid frequencies in structural building blocks,a protein secondary structure classification scheme

To study local structures in proteins, we previously developed an autoassociative artificial neural network (autoANN) and clustering tool to discover intrinsic features of macromolecular structures. The hidden unit activations computed by the trained autoANN are a convenient low-dimensional encoding of the local protein backbone structure. Clustering these activation vectors results in a unique classification of protein local structural features called Structural Building Blocks (SBBs). Here we describe application of this method to a larger database of proteins, verification of the applicability of this method to structure classification, and subsequent analysis of amino acid frequencies and several commonly occurring patterns of SBBs. The SBB classification method has several interesting properties: 1) it identifies the regular secondary structures, α helix and β strand; 2) it consistently identifies other local structure features (e.g., helix caps and strand caps); 3) strong amino acid preferences are revealed at some positions in some SBBs; and 4) distinct patterns of SBBs occur in the “random coil” regions of proteins. Analysis of these patterns identifies interesting structural motifs in the protein backbone structure, indicating that SBBs can be used as “building blocks” in the analysis of protein structure. This type of pattern analysis should increase our understanding of the relationship between protein sequence and local structure, especially in the prediction of protein structures. © 1997 Wiley-Liss, Inc.     

The interaction of capping protein with the barbed end of the actin filament

The interaction of capping protein (CP) with actin filaments is an essential element of actin assembly and actin-based motility in nearly all eukaryotes. The dendritic nucleation model for Arp2/3-based lamellipodial assembly features capping of barbed ends by CP, and the formation of filopodia is proposed to involve inhibition of capping by formins and other proteins. To understand the molecular basis for how CP binds the barbed end of the actin filament, we have used a combination of computational and experimental approaches, primarily involving molecular docking and site-directed mutagenesis. We arrive at a model that supports all of our biochemical data and agrees very well with a cryo-electron microscopy structure of the capped filament. CP interacts with both actin protomers at the barbed end of the filament, and the amphipathic helix at the C-terminus of the β-subunit binds to the hydrophobic cleft on actin, in a manner similar to that of WH2 domains. These studies provide us with new molecular insight into how CP binds to the actin filament.     

Identification of the binding site on S100B protein for the actin capping protein CapZ.

The calcium-binding protein S100B binds to several potential target proteins, but there is no detailed information showing the location of the binding site for any target protein on S100B. We have made backbone assignments of the calcium-bound form of S100B and used chemical-shift changes in spectra of 15N-labeled protein to locate the site that binds a peptide corresponding to residues 265-276 from CapZ alpha, the actin capping protein. The largest chemical-shift changes are observed for resonances arising from residues around the C terminus of the C-terminal helix of S100B and residues Val-8 to Asp-12 of the N-terminal helix. These residues are close to but not identical to residues that have been identified by mutational analysis to be important in other S100 protein-protein interactions. They make up a patch across the S100B dimer interface and include some residues that are quite buried in the structure of calcium-free S100B. We believe we may have identified a binding site that could be common to many S100 protein-protein interactions.     

Kinase partner protein interacts with the LePRK1 and LePRK2 receptor kinases and plays a role in polarized pollen tube growth

The pollen-specific receptor kinases LePRK1 and LePRK2 have localization and expression profiles that strongly suggest they play roles in pollen germination and tube growth. To identify downstream components of LePRK signaling, we used their cytoplasmic domains (CDs) as baits in yeast two-hybrid screens of a tomato pollen cDNA library. A pollen-specific protein we named kinase partner protein (KPP) interacted with the CDs of both LePRK1 and LePRK2 in yeast and in an in vitro pull-down assay, and with LePRK2 in a co-immunoprecipitation assay. KPP is a peripheral membrane protein and is phosphorylated in pollen. Pollen tubes over-expressing KPP developed balloon-like tips with abnormal cytoplasmic streaming and F-actin arrangements and plants over-expressing KPP exhibited impaired transmission of the transgene through the male. KPP-like genes are found only in plants; the 14 family members in Arabidopsis thaliana exhibit diverse expression patterns and potentially play roles in signaling pathways in other tissues.     

NMR structural characterization of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum

Cyclase-associated proteins (CAPs) are highly conserved, ubiquitous actin binding proteins that are involved in microfilament reorganization. The N-termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C-termini bind to G-actin. We report here the NMR characterization of the amino-terminal domain of CAP from Dictyostelium discoideum (CAP(1-226)). NMR data, including the steady state (1)H-(15)N heteronuclear NOE experiments, indicate that the first 50 N-terminal residues are unstructured and that this highly flexible serine-rich fragment is followed by a stable, folded core starting at Ser 51. The NMR structure of the folded core is an alpha-helix bundle composed of six antiparallel helices, in a stark contrast to the recently determined CAP C-terminal domain structure, which is solely built by beta-strands.     

GMAP-210, A cis-Golgi network-associated protein, is a minus end microtubule-binding protein

We report that a peripheral Golgi protein with a molecular mass of 210 kD localized at the cis-Golgi network (Rios, R.M., A.M. Tassin, C. Celati, C. Antony, M.C. Boissier, J.C. Homberg, and M. Bornens. 1994. J. Cell Biol. 125:997-1013) is a microtubule-binding protein that associates in situ with a subpopulation of stable microtubules. Interaction of this protein, now called GMAP-210, for Golgi microtubule-associated protein 210, with microtubules in vitro is direct, tight and nucleotide-independent. Biochemical analysis further suggests that GMAP-210 specifically binds to microtubule ends. The full-length cDNA encoding GMAP-210 predicts a protein of 1, 979 amino acids with a very long central coiled-coil domain. Deletion analyses in vitro show that the COOH terminus of GMAP-210 binds to microtubules whereas the NH2 terminus binds to Golgi membranes. Overexpression of GMAP-210-encoding cDNA induced a dramatic enlargement of the Golgi apparatus and perturbations in the microtubule network. These effects did not occur when a mutant lacking the COOH-terminal domain was expressed. When transfected in fusion with the green fluorescent protein, the NH2-terminal domain associated with the cis-Golgi network whereas the COOH-terminal microtubule-binding domain localized at the centrosome. Altogether these data support the view that GMAP-210 serves to link the cis-Golgi network to the minus ends of centrosome-nucleated microtubules. In addition, this interaction appears essential for ensuring the proper morphology and size of the Golgi apparatus.     

The actin cytoskeletal network plays a role in yeast prion transmission and contributes to prion stability

Chaperone networks are required for the shearing and generation of transmissible propagons from pre-existing prion aggregates. However, other cellular networks needed for maintaining yeast prions are largely uncharacterized. Here, we establish a novel role for actin networks in prion maintenance. The [PIN⁺] prion, also known as [RNQ⁺], exists as stable variants dependent upon the chaperone machinery for the transmission of propagons to daughter cells during cell division and cytoplasmic transfer. Loss of the Hsp104 molecular chaperone leads to the growth of prion particles until they are too large to be transmitted. Here, we isolated a unique [PIN⁺] variant, which is unstable in actin mutants. This prion loss is observed over many generations, and coincides with the detection of both high molecular weight species of Rnq1 and large visible aggregates that are asymmetrically retained during cell division. Our data suggest that the irregular actin networks found in these mutants may influence propagon number by slowly permitting aggregate growth over time, resulting in the generation of nontransmissible large aggregates. Thus, we show the potential contribution of cytoskeletal networks in the transmission of prion propagons, which parallels models that have been proposed for cell-to-cell transmission of small amyloids in neurodegenerative protein aggregation diseases.     

Formation of filopodia-like bundles in vitro from a dendritic network

We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)-activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)-coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.     

Capping and dynamic relation between domains 1 and 2 of gelsolin

Gelsolin is a protein that severs and caps actin filaments. The two activities are located in the N-terminal half of the gelsolin molecules. Severing and subsequent capping requires the binding of domains 2 and 3 (S2–3) to the side of the filaments to position the N-terminal domain 1 (S1) at the barbed end of actin (actin subdomains 1 and 3). The results provide a structural basis for the gelsolin capping mechanism. The effects of a synthetic peptide derived from the sequence of a binding site located in gelsolin S2 on actin properties have been studied. CD and IR spectra indicate that this peptide presented a secondary structure in solution which would be similar to that expected for the native full length gelsolin molecule. The binding of the synthetic peptide induces conformational changes in actin subdomain 1 and actin oligomerization. An increase in the polymerization rate was observed, which could be attributed to a nucleation kinetics effect. The combined effects of two gelsolin fragments, the synthetic peptide derived from an S2 sequence and the purified segment 1 (S1), were also investigated as a molecule model. The two fragments induced nucleation enhancement and inhibited actin depolymerization, two characteristic properties of capping. In conclusion, for the first time it is reported that the binding of a small synthetic fragment is sufficient to promote efficient capping by S1 at the barbed end of actin filaments. ©1998 European Peptide Society and John Wiley & Sons, Ltd.     

Conserved abundance and topological features in chromatin‐remodeling protein interaction networks

The study of conserved protein interaction networks seeks to better understand the evolution and regulation of protein interactions. Here, we present a quantitative proteomic analysis of 18 orthologous baits from three distinct chromatin‐remodeling complexes in Saccharomyces cerevisiae and Homo sapiens. We demonstrate that abundance levels of orthologous proteins correlate strongly between the two organisms and both networks have highly similar topologies. We therefore used the protein abundances in one species to cross‐predict missing protein abundance levels in the other species. Lastly, we identified a novel conserved low‐abundance subnetwork further demonstrating the value of quantitative analysis of networks.     

Modeling of protein binary complexes using structural mass spectrometry data

In this article, we describe a general approach to modeling the structure of binary protein complexes using structural mass spectrometry data combined with molecular docking. In the first step, hydroxyl radical mediated oxidative protein footprinting is used to identify residues that experience conformational reorganization due to binding or participate in the binding interface. In the second step, a three-dimensional atomic structure of the complex is derived by computational modeling. Homology modeling approaches are used to define the structures of the individual proteins if footprinting detects significant conformational reorganization as a function of complex formation. A three-dimensional model of the complex is constructed from these binary partners using the ClusPro program, which is composed of docking, energy filtering, and clustering steps. Footprinting data are used to incorporate constraints-positive and/or negative-in the docking step and are also used to decide the type of energy filter-electrostatics or desolvation-in the successive energy-filtering step. By using this approach, we examine the structure of a number of binary complexes of monomeric actin and compare the results to crystallographic data. Based on docking alone, a number of competing models with widely varying structures are observed, one of which is likely to agree with crystallographic data. When the docking steps are guided by footprinting data, accurate models emerge as top scoring. We demonstrate this method with the actin/gelsolin segment-1 complex. We also provide a structural model for the actin/cofilin complex using this approach which does not have a crystal or NMR structure.     

Preferential duplication in the sparse part of yeast protein interaction network

Gene duplication is an important mechanism driving the evolution of biomolecular network. Thus, it is expected that there should be a strong relationship between a gene's duplicability and the interactions of its protein product with other proteins in the network. We studied this question in the context of the protein interaction network (PIN) of Saccharomyces cerevisiae. We found that duplicates have, on average, significantly lower clustering coefficient (CC) than singletons, and the proportion of duplicates (PD) decreases steadily with CC. Furthermore, using functional annotation data, we observed a strong negative correlation between PD and the mean CC for functional categories. By partitioning the network into modules and assigning each protein a modularity measure Q(n), we found that CC of a protein is a reflection of its modularity. Moreover, the core components of complexes identified in a recent high-throughput experiment, characterized by high CC, have lower PD than that of the attachments. Subsequently, 2 types of hub were identified by their degree, CC and Q(n). Although PD of intramodular hubs is much less than the network average, PD of intermodular hubs is comparable to, or even higher than, the network average. Our results suggest that high CC, and thus high modularity, pose strong evolutionary constraints on gene duplicability, and gene duplication prefers to happen in the sparse part of PINs.