Immunology
Studies Unmask Undercover Asthma Agent
Genetics
X Inactivation Seen as Contact Sport
Child Health
Child Enrichment Program Still Pays Off After 15 Years
Education
Cut by Majors, Pitcher Winds Up at HMS
Protein Reengineered for Research and Drug Design
Small Molecules Quash Virulent Infection
Older Pathways Illuminate Newer Genetic Regulators
Women’s Work Is at the Bench and Bedside
Proceedings of the HMS Faculty Council
Partners Receives Award for Human Research
Protein Reengineered for Research and Drug Design
Research continues to improve the ability of computational design algorithms to optimize the properties of a given protein. A study in the Feb. 24 Journal of Biological Chemistry describes the successful use of such tools to devise a new intercellular adhesion molecule (ICAM-1) with enhanced binding affinity and potency.
Stuck on you. In the top image, the alpha-L I domain of the
LFA-1 integrin receptor (light gray) binds to the wild-type intercellular
adhesion molecule ICAM-1 (dark gray), and in the bottom image, it binds
with higher affinity to the Hi3 ICAM-1 mutant. The labeled segments (red) illustrate
side-chain residues of ICAM-1 that were mutated to create Hi3.
“We set out to make high-affinity ICAM-1, but we were actually more
interested in its receptor,” said Gang Song, HMS research fellow in
pathology at the CBR Institute for Biomedical Research.
ICAM-1 binds to the integrin lymphocyte function-associated antigen-1 (LFA-1) receptor to mediate inflammation and white blood cell trafficking. In 2003, co-author Timothy Springer, the Latham family professor of pathology at the CBR, and colleagues generated three-dimensional crystal structures of the binding site of LFA-1, the inserted (I) domain of the receptor’s extracellular alpha-L subunit. The structures showed three distinct conformations—open, intermediate, and closed—in a shape-shifting pathway. “These conformations happen physiologically, but we can’t differentiate them in vitro with wild-type, low-affinity ICAM-1,” said Song. A higher affinity ICAM-1 would bind more tightly and allow researchers to detect the different conformations of LFA-1.
The team used the three-dimensional crystal structure of the I-domain bound with ICAM-1 as a computational starting point for designing a high-affinity version of the ligand. Keeping the protein backbone fixed, they investigated varying the amino-acid side chains. Two different algorithms, the Sequence Prediction Algorithm (SPA) and Rosetta, suggested genetic changes expected to bring about increases in binding affinity. Researchers tested each suggested mutation, then combined those that were productive, looking for optimal modifications. “The predictions narrowed it down to a small number of substitutions, whereas random selections would have taken millions of attempts,” said Springer.
This iterative process resulted in three ICAM-1 variants, Hi1, Hi2, and Hi3, with 9-, 13-, and 22-fold increases in affinity from wild-type without losing structural integrity or compromising expression. Song is using these ICAM-1 mutants to study the cell biology of the intermediate conformations of LFA-1.
A better understanding of this shape shifting could lead to better treatments for autoimmune diseases. The success of existing drugs that target the interaction between ICAM-1 and LFA-1, such as one for psoriasis, said Springer, suggests that these ICAM-1 variants may be a useful starting point for the design of new therapeutic drugs.
Small Molecules Quash Virulent Infection
Human cytomegalovirus (HCMV) can cause severe disease in newborns and immunocompromised individuals, such as recipients of organ transplants and patients with AIDS. A new study has identified five small molecules that disrupt the replication of HCMV and produce an antiviral effect with relatively low toxicity, according to a report in the Feb. 24 issue of Chemistry and Biology.
“We think these molecules are great starting points for potentially less toxic and more effective drugs for HCMV,” said co-author Donald Coen, HMS professor of biological chemistry and molecular pharmacology.
Earlier work from Coen’s lab with herpes simplex virus (HSV) had shown that a single amino acid change in a key viral enzyme could disrupt a particular protein–protein interaction and prevent viral replication. “This [discovery] led us to believe that small molecules could do the same thing,” said Coen.
The researchers turned to HCMV, which relies on a protein–protein interaction between DNA polymerase subunits UL54 and UL44 homologous to the one they studied in HSV. First author Arianna Loregian, HMS research fellow in Coen’s lab, now at the University of Padua, discovered that a single amino acid change also disrupted the UL54–UL44 interaction. With that finding plus a crystal structure from the lab of James Hogle, the Edward S. Harkness professor of biological chemistry and molecular pharmacology, Loregian hypothesized that a small molecule could effectively plug a crevice on subunit UL44 and prevent binding with UL54.
Using a fluorescence polarization screen, Loregian analyzed nearly 50,000 small molecules to identify those capable of blocking the interaction between a UL54-derived peptide and UL44. Among the 25 molecules that specifically disrupted the interaction, only 21 were commercially available. Of those, five inhibited the interaction of UL54 and UL44 and the long-chain DNA synthesis performed by the two proteins. These molecules also had relatively low toxicity levels.
Because protein–protein interactions are so specialized, they make attractive drug targets. “But not all of them make good targets,” said Coen. The protein interfaces often involve many contact points. In some cases, the whole interaction relies on many contacts together. But in others, like those involving HCMV and HSV, a few crucial contacts make a key interaction vulnerable to disruption.
Coen and colleagues are continuing this work, focusing on pinpointing which protein the five effective small molecules are binding to and verifying that they act as their analyses predicted. They also hope to have these molecules tested in animal models. “We’d love to find a corporate partner for drug development,” Coen said.
Older Pathways Illuminate Newer Genetic Regulators
Several kinases regulating the NFAT transcription factor, a protein widely involved in vertebrate development and function, were identified using biochemical techniques. Yet one link in the chain of signals remained elusive. A study in the March 1 Nature has identified this link by using a genomewide RNAi screen for NFAT regulators in Drosophila, an organism that itself lacks NFAT. This novel approach crosses evolutionary boundaries, leveraging pathways conserved between Drosophila and vertebrates to study a protein that emerged more recently in evolution.
To verify the feasibility of a Drosophila RNAi screen for identifying regulators of vertebrate NFAT, Anjana Rao, HMS professor of pathology at the CBR Institute for Biomedical Research, and research associate Yousang Gwack introduced an NFAT–GFP fusion protein into Drosophila S2R+ cells. The cells, when stimulated to raise intracellular free calcium levels, moved NFAT through the same cycle observed in mammalian cells. Starting in a resting state, in which NFAT is phosphorylated in the cytoplasm, an external stimulus triggered calcineurin to dephosphorylate NFAT and translocate it to the nucleus. Removing the stimulus reversed the flow.
Previous research from the Rao lab had described three phosphorylated regions on NFAT and had identified kinases for two of these three regions. When standard biochemical methods failed to find the third, Rao opted for the Drosophila screen.
Once the screen identified Drosophila kinases, Sonia Sharma, HMS research fellow in pathology, tested human homologues for their ability to phosphorylate NFAT. Only the kinase DYRK countered the dephosphorylation of NFAT, yielding fully phosphorylated NFAT in the cell. The investigators had found their missing kinase—and something more. Gain- and loss-of-function experiments revealed a multistage cascade of signals that deactivate NFAT. The process starts in the nucleus, when DYRK1A phosphorylates the NFAT SP3 motif. This primes the previously identified kinases, CK1 and GSK3, to phosphorylate the remaining two motifs, SP2 and SRR1, respectively.
Many transcription factors have multiple phosphorylation sites, said Rao, but NFAT exhibits “discontiguous” priming—the serines phosphorylated by CK1 and GSK3 are not adjacent to the priming site. Rao is investigating the possibility that the early steps in the cascade cause a conformational change that facilitates the later steps. “We should be looking for this more complex picture in other transcription factors,” she said. “Many proteins are regulated by multiple kinases, possibly in multiple steps involving discontiguous priming.”
This study validates the use of Drosophila screens for investigating complex mammalian signaling pathways. Many screens are publicly available through the Drosophila RNAi Screening Center at HMS, which opened in May 2003 (see Focus, Jan. 23, 2004).