Research
My lab studies receptor-ligand interactions and signal transmission across membranes. We use a wide range of structural, cell biological, and single molecule techniques to answer important questions relevant to immunology, hemostasis, mammalian biology, and human disease. A common theme throughout our research is how force interacts with protein conformational change to activate integrins, von Willebrand factor, proteins of the transforming growth factor-beta family, and adhesins on malaria sporozoites. Some areas that currently fascinate us follow.
Integrins | von Willebrand factor | Malaria | TGF-β Family of Ligands | Therapeutics
Integrins
How are conformational signals transmitted across membranes in integrins, over long distances between the distal ligand-binding domains and cytoplasmic domains, and how is integrin activation coordinated with the actin cytoskeleton to mediate cell migration? How do G proteins and kinases activate integrins (inside-out signaling) so they can bind to extracellular ligands and cytoskeletal proteins, to coordinate adhesion and directional cell migration and mediate inside-out and outside-in signaling? How is integrin activation confined to lamellipodia? We have proposed that a mechanism for coordinating binding to ligands outside the cell and the cytoskeleton inside the cell is inherent in integrin structure and the allosteric pathways that relate the bent - closed headpiece; extended- closed headpiece; and extended – open headpiece conformations. Cell biological experiments are required to test this hypothesis.
The three overall integrin conformational states. (A) The bent conformation has a closed headpiece and is low affinity. Extension at the α- and β-knees releases an interface between the headpiece and lower legs and yields an extended-closed conformation also with low affinity. Swing-out of the hybrid domain at its interface with the βI domain is connected through the βI α7-helix to rearrangements at the βI interface with the β-propeller domain that greatly (~1,000-fold) increase affinity for ligand in the extended-open conformation. Similar interdomain rearrangements in αI integrins result in activating a binding site for an internal ligand, Glu310 in αL, which pulls down the αI α7-helix to activate a similar increase in affinity (~1,000 to 10,000-fold) of the αL I domain for the ligand ICAM-1. Although the integrin headpiece has highly preferred closed and open conformations, the lower β-legs are highly flexible, and thus we speak of “overall” conformational states. This is symbolized by the dashed lower β-leg. Therefore, only very large separations between α and β TMD, such as induced by lateral motion of β when its cytoplasmic domain is associated with the actin cytoskeleton, can be transmitted through the floppy β-leg to stabilize the high-affinity, open headpiece conformation. (B) Crystal structure of the “closed” α5β1 integrin.
How do representative members of the integrin family work? This family is quite diverse, and we study representative integrins including LFA-1, Mac-1, αXβ2, α4β1, and α4β7on leukocytes, αIIbb3 on platelets, and the αV integrins that bind and activate TGF-β. To gain a broad overview, we may extend to other subfamilies including those that recognize collagen and laminin.
How can integrins, depending on their activation state, mediate transient adhesion that supports rolling, or alternatively, firm adhesion, in postcapillary venules? This question is being addressed with integrins α4β1, α4β7, and their ligands MAdCAM-1, and VCAM-1, which mediate both lymphocyte homing in the vasculature and cell adhesion and immune responses in tissues. Furthermore, we are examining how small drug-candidate molecules and antibodies in the clinic for asthma, multiple sclerosis, and inflammatory bowel disease bind to these integrins.
We have initiated single molecule laser tweezer and atomic force microscopy measurements for understanding the mechanobiology of cell adhesion and diverse physiologic processes including hemostasis and thrombosis. How can adhesion molecules such as integrins and selectins resist substantial forces that should break receptor-ligand noncovalent bonds? Does the fact that many adhesion receptors have high affinity conformations that are more extended than the low affinity conformation along the cell attachment site - ligand binding site axis give them a mechanical advantage in resisting force? Can we measure this using novel receptor-ligand constructs with laser tweezers or the atomic force microscope?
von Willebrand factor
One new area of research in the lab is von Willebrand factor and mutations that cause the important bleeding diathesis, von Willebrand disease. A fundamental question we are addressing is whether von Willebrand factor senses shear in the bloodstream and activates hemostasis because extension reveals otherwise hidden receptor binding sites? We are pursuing single molecule experiments, functional assays, electron microscopy, and crystallography to understand the complex mechanobiology of VWF, which is the largest soluble protein in the body and functions as a shear sensor in the vasculature to arrest arteriolar bleeding. The conformation of VWF, its binding to ligands on the vessel wall and on platelets, and its cleavage by ADAMTS13 are regulated by shear.
The VWF A1 and GP1bα ReaLiSM construct and change in extension upon unbinding and rebinding. (A) Schematic of the ReaLiSM construct and laser tweezers. (B) Successive cycles of stretching (black traces) and relaxation (red traces) with unbinding and rebinding events arrowed, with the R1306Q construct at 40 nm/s. (C) Fits to worm-like chain model showing the persistence length (PL) and contour length (CL). Bars show SD for each force bin.
Malaria
A second new area is malaria. We posit that the structures of important vaccine targets will reveal both interesting biology, and enable rational development of vaccines. These vaccines will focus the immune response on conserved epitopes, and avoid masking by polymorphic epitopes that pathogens often use to evade immune responses. Proteins on sporozoites are candidates for vaccines that prevent infection. We are interested in determining the structure of the sheath that surrounds sporozoites, its main component the circumsporozoite protein, and TRAP, which mediates sporozoite gliding motility and invasion of mosquito and vertebrate host cells. Proteins on gametocytes are targets for vaccines that prevent Plasmodium fertilization and infection in mosquitoes, and prevent transmission to other humans. These proteins include a family unique to Apicomplexans with 6-Cys domains, and HAP2, a putative gamete fusogen conserved in many eukaryotic phyla.
Model of TRAP activation. (A) In the absence of ligand and tensile force, the closed conformation with a flexible orientation between the VWA and TSR domains is favored. (B) When an immobilized ligand binds to TRAP through the MIDAS, the tensile force exerted by the parasite actomyosin apparatus (magenta ovals) will be resisted. TRAP will then be elongated and straightened along the force vector. The elongational force is on pathway with TRAP extension through activation of the VWA domain and formation of the extensible β ribbon. Hence the extended open conformation with putative high-affinity is favored. (C) Structure of Plasmodium vivax 25-283 fragment in the open conformation. VWA = von Willbrand factor type A, TSR = thrombospondin type I repeat.
Malaria Publications
Transforming growth factor beta family of ligands
A third new area is TGF-β signaling, which regulates development, wound healing, immune responses, and tumor-cell growth and inhibition. Latent TGF-β and receptors for active TGF-β are ubiquitous; it is activation of TGF-β in the extracellular space that limits activity. We have determined a structure of the latent TGF-β1 procomplex (pro-TGF-β1), which reveals how the latency associated peptide (LAP), a 250 residue prodomain, forms a ring that shields the 110-residue growth factor from receptor-binding and participates in its activation through interactions with αV integrins and latent TGF-β binding proteins (LTBPs). We also have studied how a different cell surface protein called GARP becomes disulfide linked to latent TGF-β, and presents it for activation by integrins αVβ6 and αVβ8 that are expressed on immune cells, and bind to an RGD motif in LAP. We believe that yet another cell surface molecule that presents latent TGF-β remains to be discovered.
Structure and activation mechanism of pro-TGF-β1. (A) Pro-TGF-β1 adopts a cross-armed, ring-like conformation. The prodomain arm domains are disulfide-linked in the bowtie region, and the prodomain straitjacket regions (latency lasso, fastener, and α1 helix) surround the growth factor dimer and block binding of TGF-β receptors. Red arrows show the directions of forces during activation by integrins. (B) Tensile force applied to the RGD motifs in the shoulders by αV integrins attached to the actin cytoskeleton will be resisted at the opposite end of the ring, where the Cys 4 residues (A) in the straitjacket are disulphide-linked to latent TGF-β binding protein (LTBP), which is tightly associated with the ECM. The applied force unfastens the straitjacket and releases TGF-β from the prodomain for receptor binding.
We are now interested in understanding how other proteins of the 33-member TGF-β family are activated, particularly TGF-β2, which lacks the RGD integrin-binding motif in its LAP sequence. More recently, we have solved the structure of the closely related bone morphogenetic protein 9 (BMP9) procomplex, which adopts a markedly different conformation than pro-TGF-β1 that is consistent with its non-latency. These studies are further uncovering the tremendous functional and structural diversity within the TGF-β family, and a wide range of projects, all the way from knockout mice to structure determination, are in the offing.
Therapeutics
Finally, we always strive to make connections between basic research and disease. In the past, we have found inherited defects of integrins in leukocyte adhesion deficiency, ICAM-1 as the cellular receptor for rhinovirus, and SDF-1 as the natural ligand for the HIV coreceptor CXCR4. Our discoveries of LFA-1 and LFA-3 resulted in the drugs efalizumab (Raptiva, LFA-1 antibody, Genentech) and alefacept (Amevive, LFA-3-Fc fusion protein, Biogen). We are currently developing and characterizing antibodies specific for the activated conformation of integrin I domains as therapeutics for autoimmune disease. Our work has important implications for development and improvement of therapeutics directed to many receptors.