Biological Chemistry and Molecular Pharmacology
We work on 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.
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.
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? Does von Willebrand factor sense shear in the bloodstream and activate hemostasis because extension reveals otherwise hidden receptor binding sites?
One new area of research in the lab is von Willebrand factor and mutations that cause the important bleeding diathesis, von Willebrand disease. 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.
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 the motility protein TRAP. 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.
A third new area is TGF-β signaling, which regulates development, wound healing, immune responses, and tumour-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.
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.
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.
We work on 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.
Timothy A. Springer received his B.A. in Biochemistry from University of California in 1971, his Ph.D. in Molecular Biology and Biochemistry from Harvard in 1976, and did a fellowship with Cesar Milstein in Cambridge, England. He began as Assistant Professor on the Quad at HMS in 1977 and has been here ever since, although his institution has changed several times. Since 1989 Springer has been Latham Family Professor. He currently is Professor of Biological Chemistry and Molecular Pharmacology and of Medicine at HMS and in the Program of Cellular and Molecular Medicine, and in the Division of Hematology, Department of Medicine at Children’s Hospital.
Springer discovered with monoclonal antibodies, then cloned and functionally and structurally characterized, many of the adhesion receptors in the immune system. He was the first to demonstrate that lymphocytes and leukocytes had adhesion molecules. His work on these receptors has advanced to characterizing their interactions and allosteric transitions by x-ray crystallography, electron microscopy, and laser tweezers force spectroscopy.
He discovered the lymphocyte function-associated (LFA) molecules, the intercellular adhesion molecules (ICAMs), and the first subfamily of integrins. He discovered that LFA-1 bound to ICAM-1, that LFA-2 (CD2) bound to LFA-3, and that blocking either of these adhesion pathways could block antigen recognition by T lymphocytes. These two pathways were the first examples of like-unlike cell adhesive recognition in biology. These discoveries directly led to the development and FDA approval of efalizumab (Raptiva, Genentech) - an antibody to LFA-1, and Alefacept (Amevive, Biogen) - the LFA-3 ectodomain fused to Fc, both for plaque psoriasis.
Springer later discovered the three step paradigm for leukocyte diapedesis: 1) rolling adhesion of leukocytes on the vessel wall through a translating zone of selectin-carbohydrate adhesion; 2) activation of G protein-coupled receptors on the leukocyte by chemoattractants presented by vascular endothelium; and 3) activation of integrin adhesiveness for CAMs on endothelium, which mediates firm adhesion and leukocyte migration through the vessel wall. The use of different molecular digits in each step creates area codes that govern which particular subset of leukocytes or lymphocytes will emigrate from the bloodstream to a particular subset of inflammatory signals. This together with the realization that blocking any of these steps could completely block emigration led Springer to found LeukoSite in 1993. In 1996 LeukoSite published that an antibody to integrin a4b7, a lymphocyte homing receptor for mucosal tissues, rapidly resolved colitis in non-human primates. This antibody, vedolizumab (Entyvio, Takeda) was approved for moderate to severe ulcerative colitis and Crohns disease in 2014. Springer recruited to the SAB Herman Waldmann, the creator of the antibody CAMPATH-1 (Alemtuzumab). Waldmann arranged to license CAMPATH-1 to LeukoSite in 1997, which was approved by the FDA for B cell chronic lymphocytic leukemia in 2001. CAMPATH was later acquired by Genzyme, and reintroduced as Lemtrada, which was approved for multiple sclerosis in 2013. LeukoSite went public in 1998 and acquired ProScript and with it the proteasome inhibitor bortezimib in 1999, which was approved for multiple myeloma in 2003 (Velcade, Millenium Pharmaceuticals). LeukoSite was acquired by Millenium in December 1999 and as 35% of Millenium was valued at $3 billion by 2001.
Springer’s academic interests now focus on how protein conformational change together with tensile force activates integrins, von Willebrand factor, the transforming growth factor-b family, and adhesins on malaria sporozoites, and discovering new binding partners. Springer has over 500 publications, a Hirsch index of 147, and over 30 patents. He is a member of the National Academy of Sciences and his honors include the Crafoord Prize, the American Association of Immunologists Meritorious Career Award, and the Stratton Medal from the American Society of Hematology.
Springer is an investor in Selecta Bioscience since its B round and a founding investor in Moderna Therapeutics. He is a Resident Professor at Pfizer. He is founder and investor in Scholar Rock and in Morphic Rock Therapeutics, and board member and lead angel of Ab Initio Biotherapeutics. As a philanthropist, Springer has endowed Chairs at Harvard Medical School and Children’s Hospital, and is on the Children’s Hospital Boston Board of Trust.
Current projects include a non-profit to advance entrepeneurship and innovation in protein therapeutics and open-source antibodies and small molecules. Next is a dual-acting malaria vaccine, to both protect humans from being infected, and to prevent mosquitos from transmitting infection.
For the most up to date list of publications, please use this link.