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STRUCTURAL BIOLOGY Molecular Jumping Jack Shows Off MovesIntegrin's Technique Underlies Blood Clotting, Cancer Spread, Other Pivotal Events When cells stick together they are held fast by integrins. The adhesive interactions are fine-tuned according to the affinities and regulatory quirks of each integrin species, and they lie dormant until activated by specific signals. A report from HMS researchers in the April Nature Structural Biology offers one model of how the proteins literally spring into action.
Right, the integrin molecule snaps open like a switchblade from the low-affinity state, according to Junichi Takagi, Timothy Springer, Stephen Blacklow, and Natalia Beglova (l to r, above). At the "epicenter" of movement are the epiderminal growth factor (EGF)-like domains 2 and 3, located in the extreme bend of the protein in its resting state. The figure below, right, shows a surface representation of the molecule superimposed on a ribbon trace of the backbone. By solving the solution structure of these domains, the researchers could position amino acid epitopes for activating monoclonal antibodies (below, pink spheres) and amino acid residues that restrain activation (black spheres) and show that they were masked or buried in the resting state. (Photo by Pam Murray; Images adapted from originals from Timothy Springer) Co-lead authors Natalia Beglova and Stephen Blacklow, HMS assistant professor of pathology at Brigham and Women's Hospital, together with Junichi Takagi and Timothy Springer, the Latham Family professor of pathology at the Center for Blood Research, reported a nuclear magnetic resonance (NMR) solution structure of what Springer calls the integrin "epicenter": a small yet key region of the molecule that undergoes a dramatic conformational change upon activation. Its movement underlies the molecular mechanisms that among other things allow blood to clot, organs to develop, cells to metastasize, and lymphocytes to home to infected tissue. TransformersSpringer is quick to summarize his fascination with integrins: "They change shape. Rapidly."This is the essence of the integrin character, probably unique to cell surface proteins: integrins change form, and this affects the vigor with which they latch onto a substrate. At least two states are recognized, a tight binding or high-affinity conformation and one displaying a weaker, low affinity. And the protein can flick back and forth between them. But the precise details of this transition were obscure. Like Eadweard Muybridge's photographs of a galloping horse, it has been necessary to construct "snapshots" of the molecule, frozen in various states of activation, to reveal its motion. A major advance last fall was the partial (eight of 12 domains) solution of an integrin x-ray crystal structure by a team led by M. Amin Arnaout, HMS professor of medicine at Massachusetts General Hospital. The new study fills in some of the missing pieces, allowing more definitive conclusions about the molecule's activation state during movement. From Form to FunctionIntegrins are Frankenstein-like molecules: their two subunits are cobbled together from parts--some homologous to bits of other proteins--to form a hulking whole. An oversized head rests on heterodimer legs that cross the cell membrane and end in short cytoplasmic tails. Grafted onto the head in some integrins is an I (inserted) region; all have an I-like domain. These critical parts house the "jaws" that clamp onto cell surface molecules and the extracellular matrix and hold the cell fast.Structural studies reveal these choppers as a flat sheet surrounded by six helices. The transition between high and low affinity means major shifts along the polypeptide backbone: loops reshape and helices slip. But in terms of movement during activation, this may be only the final shudder because when activation starts inside the cell, the beast only bites in response to a "pull" on its tail. Integrins can transmit signals across the membrane in both directions--yet another unique feature of the molecule. In addition to being activated by ligand, a process that works from outside the cell to the inside for most cell surface receptors, they also undergo inside-out activation. Lymphocyte integrins, Springer points out, provide the best example. As these immune cells pass through narrow blood vessels, they track chemical signals to reach a site of inflammation. Such signals, perceived by traveling leukocytes, activate integrins from inside through the tail. In recent years, with the goal of understanding how shape-shifting throughout the molecule influences activation, the Springer lab has taken the integrin apart and put it back together again with cytoplasmic tails hobbled or tangled in coils, or with parts engineered to mutually repel. And with surgical precision, they have replaced amino acids in the backbone with others to freeze the protein in high or low affinity conformations. A rudimentary concept of inside-out activation emerged. The cytoplasmic tails spring apart when the integrin is activated, and this motion is transferred to the legs, which also undergo a change in conformation. But no one realized just how striking this change might be until a crystal structure of the integrin molecule was completed. In an independent study, Arnaout and his colleagues unveiled an integrin species, alpha-V beta-3, bowed in a bent configuration, with its head tucked up against its legs, facing the cell surface (Focus, Sept. 14, 2001). "I was surprised, like everyone else," said Springer. "No one expected it." Still, due to insufficient electron density in parts of the 3.1 angstrom structure, portions of the integrin were not resolved. Positioned in this hazy area were amino acid residues the Springer lab had determined to be critical for conveying movement along the integrin backbone. Buried TreasureUsing chemical analysis, NMR spectra, and the DNA sequences for structural homologies, Beglova and Blacklow worked with Takagi and Springer to figure out the organization of the molecule in the critical region. They found the functionally important domains wedged into the extreme bend connecting the head and legs.With the additional structure, the wealth of dissection and mapping data could be applied to predict integrin movement. Residues that lock the molecule in the low-affinity state were in the interface between the two subunits. And those amino acid residues that bind monoclonal antibodies when an integrin is in its high-affinity state were masked or buried in the crevice in the bent conformation (see figure, page 3). The authors concluded that the bent conformation represented the low-affinity integrin structure, contrary to the conclusion of the previous study. And they predicted that a dislocation of almost unprecedented magnitude (only the flu hemagglutinin fusion protein displays such movement) would unmask the activation epitopes. "The bent conformation represents the resting (low-affinity) conformation of the integrin," said Blacklow. "Its ligand-binding head is directed toward the cell membrane, concealed from the extracellular matrix. The switchblade-like opening would unmask the buried epitopes, which become exposed upon conversion to the high-affinity form." But the model provokes new questions. For example, how does the curled up, low-affinity form mediate signaling from outside to inside? "The story of integrin movement will continue to evolve," Springer promised. --Anne Mahon |
