Actin dynamics play important roles in eukaryotic cellular processes ranging from motility, division, and polarization to bacterial infection. To achieve complex cellular behaviors, actin rearrangements must be regulated both spatially and temporally and integrated with functions including gene expression and membrane trafficking. The rapid nucleation of new filaments underlies many actin-based processes. Since nucleation is inherently slow, specialized machineries have evolved to accelerate it in vivo (Figure 1). Most prominent among these are Arp2/3 complex and the formin proteins. The Rho-family GTPases—Cdc42, Rac, and Rho—are prominent players in the transmission and integration of signals that control the cytoskeleton, and they exert many of their functions by acting on these two nucleators.
My lab takes a multidisciplinary approach to understand mechanisms of Rho GTPase signaling that control Arp2/3 complex and formins. We study the basic structural, thermodynamic, and kinetic properties of these proteins and how interactions between them drive conformational changes that alter their activities. We also use novel engineered reagents that report on and control rapid signaling events in vivo to study how these physical properties guide information transfer in signaling pathways in the cell.
Signaling to Arp2/3 Complex
The GTPases Cdc42 and Rac communicate with the actin cytoskeleton through their interactions with members of the Wiskott-Aldrich syndrome protein (WASP) family, a group of proteins that integrate GTPase and other signals to control Arp2/3 complex. Cdc42 interacts directly with the archetypal member of the family, WASP, and with the homolog neuronal WASP (N-WASP). Rac controls the more distantly related WAVE proteins by interacting with components of a large WAVE-containing assembly. WASP proteins are characterized by a conserved C-terminal VCA domain that stimulates the actin-nucleating activity of Arp2/3 complex. In WASP and N-WASP this domain is inhibited by intramolecular binding of an N-terminal regulatory element, the GTPase-binding domain (GBD). Interaction of the GBD with Cdc42 physically displaces the VCA, leading to activation toward Arp2/3 complex and establishing a signaling pathway from the GTPase to the cytoskeleton.
Our early work focused on the activation of WASP by Cdc42. Using nuclear magnetic resonance (NMR) spectroscopy, we solved the structures of autoinhibited WASP and of the WASP GBD in complex with Cdc42. The former structure revealed that the GBD sequesters residues of the VCA needed for Arp2/3 activation, explaining the structural basis of autoinhibition. Comparison of the two structures showed that Cdc42 activates WASP by destabilizing its folded autoinhibited domain. We also developed a quantitative thermodynamic model based on this structural mechanism that predicts WASP's hydrogen exchange behavior, Cdc42-binding affinity, and activity toward Arp2/3 complex. We have also demonstrated that efficient phosphorylation and dephosphorylation of WASP in the GBD are both contingent on binding to activated Cdc42. The requirement for contingency in both phosphorylation and dephosphorylation enables long-term storage of information by WASP following decay of GTPase signals.
More recently we showed that WAVE activity is controlled through an analogous mechanism. In cells, WAVE is incorporated constitutively into a pentameric WAVE regulatory complex (WRC) containing Pir121/Sra1, Nap1/Hem1, Abi, and HSPC300/Brick. We reconstituted the WRC from recombinant human and fly components and showed that it is inactive toward Arp2/3 complex. Rac activates the assembly in a GTP-dependent manner, probably by dissociating the VCA portion of WAVE from inhibitory elements in Pir121 and/or Nap1. Thus, WAVE proteins are controlled by the intermolecular equivalent of WASP autoinhibition. We are working to discover and characterize other WRC ligands and covalent modifications to learn how the assembly integrates multiple inputs to control Arp2/3 complex. We also are working to determine the three-dimensional structure of the WRC in different activity states by crystallography and electron microscopy. Finally, we have initiated studies of the function, regulation, and structure of the WASH proteins, WASP relatives that, like WAVE, appear to function within large multicomponent complexes. Our long-term goal is to elucidate the general principles by which members of the WASP family receive and transmit signals to control actin assembly.
We have also recently discovered a previously unrecognized mechanism, superimposed upon allostery, that controls the activity of WASP-family members toward Arp2/3 complex. That is, dimerization increases the affinity of active WASP species for Arp2/3 complex by up to 180-fold, greatly enhancing actin assembly. Dimeric WASP proteins bind two distinct sites on Arp2/3 complex, forming an assembly with 2:1 stoichiometry. This finding explains a large and apparently disparate set of observations under a common mechanistic framework. These include WASP activation by the bacterial effector EspFu and a large number of SH3-domain proteins, the effects on WASP of membrane localization/clustering and assembly into large complexes, and cooperativity between WASP and WAVE. Allostery and dimerization act in hierarchical fashion, enabling WASP/WAVE proteins to integrate different classes of inputs to produce a wide range of cellular actin responses.
This discovery has given rise to several new areas of investigation in our lab. First, we are working to discover how dimeric WASP proteins interact with Arp2/3 complex and whether the two ligands could have different functions during actin assembly in vitro and in vivo. We are also studying how dimeric WASP proteins affect other Arp2/3 ligands, including the cortactin and coronin proteins, to understand how filament networks evolve over time in the cell. Finally, one prediction of our model is that higher-order oligomerization of WASP proteins should enhance their activity and potentiate their stimulation by allosteric activators such as Cdc42. Thus, we are using a combination of computational and biochemical approaches to study how WASP oligomerization is controlled, as well as how oligomerization contributes to actin assembly during processes including bacterial pathogenesis and cell adhesion.
Actin Regulation by Formins
The formin proteins, the second major actin nucleation machinery, control production of unbranched filament arrays. Many formins are downstream targets of Rho GTPases and mediate GTPase effects on actin dynamics in structures such as the cytokinetic ring and stress fibers. The conserved formin homology 2 (FH2) domain nucleates new filaments, binds tightly to filament barbed ends, and inhibits capping proteins, but paradoxically permits rapid addition and loss of actin monomers, leading to its description as a processive cap.
We solved the crystal structure of the Bni1p FH2 domain in complex with actin. The structure reveals that the FH2 domain binds actin monomers in an orientation closely resembling a short-pitch actin filament, suggesting nucleation occurs through a templating mechanism. Functional studies have led us to a model for processive capping in which the FH2 domain exists in a dynamic equilibrium at the barbed end between a blocked configuration and an open configuration that differ in the relative orientation of the two halves of the FH2 dimer. Interconversion between these two states is required for barbed-end elongation and shrinkage in the presence of bound FH2 domain.
Our current work on formins is in several areas. First, we have initiated single-particle electron microscopy investigations, in collaboration with Masahide Kikkawa (Kyoto University) and Akihiro Narita and Yuichiro Maeda (both of Nagoya University), to image directly the FH2 domain attached to the filament. Using a combination of wild-type and mutant proteins, we hope to capture the FH2 domain in its different functional states, and ultimately to understand how the energetics of these states controls the rates and persistence of formin-mediated filament growth and shrinkage. Second, we are pursuing structural and biochemical analyses of other formins to understand how their FH2 domains are inhibited by intramolecular interactions and activated by ligands. Finally, we are planning single-molecule fluorescence studies of formins bound to the ends of actin filaments to test key dynamic features of our model for processive capping.
Combinatorial Autoinhibition in Multidomain Signaling Proteins: The Guanine Nucleotide Exchange Factor Vav
Autoinhibition in multidomain systems is typically achieved through a basic active-site repression mechanism whose energetics is modulated by combinations of additional contacts involving other domains. These interactions both suppress activity in the basal state and provide mechanisms of integrating multiple inputs to achieve signaling specificity in vivo. The structural construction of autoinhibited systems suggests that activators function by recognizing poorly populated excited states. Thus, the internal dynamics of such systems and their modulation by the domain-domain contacts that modulate autoinhibitory energetics, likely play a key role in the regulatory process.
We are studying these issues in the protein Vav, a multidomain guanine nucleotide exchange factor (GEF) for Cdc42 and Rac. Our early work revealed that autoinhibition in Vav is mediated by an N-terminal helical extension from the catalytic Dbl homology (DH) domain that folds back into the active site. A key regulatory tyrosine in this helix is buried in the active site, and we showed by NMR that its phosphorylation by upstream Src-family kinases leads to unfolding of the helix and release from the DH domain. We also discovered that phosphorylation of the buried regulatory tyrosine is greatly accelerated by prior, rapid phosphorylation of exposed additional tyrosines in the Vav N terminus, which allows SH2-mediated docking of the kinase.
Activation of Vav must occur through an excited state since the regulatory tyrosine on its inhibitory helix is buried in the DH active site in the ground state structure. In full-length Vav, autoinhibition appears to be enhanced through interactions of calponin homology, pleckstrin homology, and zinc finger domains that flank the helix-DH regulatory core. Our future work will examine the role of dynamics in Vav regulation, and how dynamics and energetics of autoinhibition are altered by domain-domain interactions. This will be accomplished by comparing phosphorylation kinetics, GEF activity, and NMR measures of internal micro- to millisecond timescale dynamics across a series of Vav constructs with varying component domains. These thermodynamic analyses will be married with structure determination of the multidomain systems. Our work will help establish general structural and energetic principles by which autoinhibited systems can be generated, function, and evolve.
Genetically Encoded Photoswitchable Proteins
During processes such as cell migration, cytoskeletal architecture changes in seconds in a highly localized fashion. The signaling pathways that control the cytoskeleton must similarly evolve rapidly and with high spatial specificity. A mechanistic understanding of such pathways requires the ability to reversibly perturb them on a timescale of seconds in a spatially defined manner. Such manipulations are impossible with existing technologies, outside of pathways that are susceptible to natural light-activatable molecules (e.g., channelrhodopsin). To fill this need we are working toward general approaches to engineer genetically encoded proteins whose activities can be controlled by light in live cells. These reagents are based on chimeras between mammalian signaling proteins and plant photoreceptors, including phytochromes (which respond to red and far-red light) and light-oxygen-voltage (LOV) domains (which respond to blue light). We hope to develop rapid methods to create such reagents and the necessary tools to use them in cells and perhaps animals. These proteins will provide the speed, reversibility, and spatial precision necessary to study the signaling dynamics that control cytoskeletal dynamics. Photoswitchable proteins could have wide utility in industrial, technological, and basic science applications.