My research has focused on understanding how molecules interact to generate function in complex biological machines, such as those involved in cell movement and the transport of material into and out of the cell nucleus. I do this by determining the structure of the molecules; the nature of the interactions between them; and how these interactions generate biological function.
Initially I studied skeletal muscle with Hugh Huxley and explored how interactions between tropomyosin, troponin and actin control contraction. I then studied the structure of thick filaments in a range of vertebrate and invertebrate muscles and used EM and protein engineering to define the molecular basis of the packing of myosin tails in thick filament shafts. A complementary study looked at the structure and assembly of intermediate filaments and nuclear lamins. My interests broadened to encompass the cytoskeleton and amoeboid cell motility. To address the molecular mechanism of cell locomotion, Tom Roberts (Florida State University) and I developed a novel motile system based on nematode sperm. Although these sperm crawl like amoeba, they have a cytoskeleton built from the 14 kD major sperm protein (MSP) instead of actin. The similarity of the motility of these sperm to actin-containing amoeba is so striking that they must use the same or very similar molecular mechanisms, but the simplicity and specialization of the nematode system is a considerable advantage in determining the fundamental molecular mechanism of amoeboid motility. We have established the structure of MSP filaments, together with the crystal structure of the protein and of its helical polymer. An in vitro motility system was developed that identified vectorial filament assembly and bundling of filaments into networks as key components of the locomotion machinery and lead to a “push-pull” model of amoeboid cell motility. In parallel with this work, I also worked on the structure and function of a range of actin bundling proteins to extend the work to more general cell motility.
My other major interest is the structure and function of the nuclear envelope, especially related to nucleocytoplasmic transport and nuclear assembly. My work has concentrated on establishing key aspects of the molecular mechanism of nuclear transport by determining the structure of the central proteins and complexes involved and showing how interactions between them generate function. Nuclear protein import is the best characterized transport pathway and is facilitated by carrier proteins called “karyopherins” that bind cargo in the cytoplasm and mediate its translocation through nuclear pores to the nucleus, after which the cargo is released and the carriers recycled to the cytoplasm to participate in another import cycle. My group has determined the structure of complexes involved in translocation through the pores, dissociation in the nucleus, and recycling. This structural information was then used to engineer mutant proteins that showed how each interaction contributes to transport. Translocation of cargo:carrier complexes though nuclear pores is facilitated by proteins (“nucleoporins”) that line the transport channel and which contain characteristic FG (phenylalanine-glycine) sequence repeats. My group established the structural basis of the interactions between carriers and FG-nucleoporins and engineered mutants that established the critical function of this interaction in mediating translocation. However, interactions with FG-nucleoporins function primarily to equilibrate cargo:carrier complexes between the nucleus and cytoplasm and transport is driven by disassembly of this complex in the nucleus, thereby preventing return of the cargo to the cytoplasm. Both dissociation of the import complex and also the recycling of the karyopherins to the cytoplasm require the Ran GTPase. Ran is charged with GTP in the nucleus that is then hydrolyzed to GDP in the cytoplasm (providing the energy that powers transport), after which Ran it is recycled to the nucleus by NTF2. My group determined the structural basis for how the nucleotide state of Ran controls the interactions between karyopherins and their cargoes, both in the nuclear import complex disassembly that drives transport and in transport factor recycling. The crystal structures of the Cse1-based nuclear export complex and of full-length importin-b complexed with RanGTP, coupled with structure-based engineered mutants, showed how Ran binding controls the affinity for cargoes by exploiting the inherent flexibility of karyopherins. These studies also indicated how energy is stored by distorting the conformation of these molecules and lead to a spring-loaded mechanism for substrate release that enabled high specificity to be modulated by small energy changes. In this way the considerable energy associated with an extensive and thus specific interaction interface could be balanced to a large extent by internal strain associated with distorting the karyopherins by binding each partner, thereby enabling the comparatively small amount of energy associated with GTP hydrolysis to effect cargo release or binding. In addition, my group showed how specific nucleoporins accelerate import complex disassembly and karyopherin recycling at the nuclear face of the pores to enable the high rates of transport seen in vivo to be achieved. This work is now being extended to establish the mechanism by which mRNA is exported, together with delineating how export is co-ordinated with transcription and nuclear mRNA processing. The yeast Zn-finger protein Nab2 is important in determining polyA tail length and compaction of mRNPs before nuclear export and the crystal structure of a complex between three of its Zn fingers and A11G RNA has provided an explanation for how these functions are mediated. In yeast, transcription, processing and export are coordinated by complexes such as TREX-2 that facilitate the localization of many actively-transcribing genes to nuclear pores (referred to as “gene gating”). My group determined the structure of the TREX-2 Sac3:Cdc31:Sus1 component that mediates tethering to the pores in yeast as well as the analogous system that contributes to mRNA export in mammalian cells. Most recently, the structure of the distal region of TREX-2 (containing Thp1, Sem1 abd Sac3) has been established and shown to have a novel nucleic acid function that facilitates assembly of export-competent mRNA particles. The function of a range of other macromolecular complexes in trafficking is currently being investigated together with the molecular interactions that generate mRNA nuclear export and the function of the TREX-2 complex in integrating mRNA nuclear export with preceding steps in the gene expression pathway.