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Dive into the research topics where Amy E. Keating is active.

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Featured researches published by Amy E. Keating.


Nature | 2009

Design of protein-interaction specificity gives selective bZIP-binding peptides

Gevorg Grigoryan; Aaron W. Reinke; Amy E. Keating

Interaction specificity is a required feature of biological networks and a necessary characteristic of protein or small-molecule reagents and therapeutics. The ability to alter or inhibit protein interactions selectively would advance basic and applied molecular science. Assessing or modelling interaction specificity requires treating multiple competing complexes, which presents computational and experimental challenges. Here we present a computational framework for designing protein-interaction specificity and use it to identify specific peptide partners for human basic-region leucine zipper (bZIP) transcription factors. Protein microarrays were used to characterize designed, synthetic ligands for all but one of 20 bZIP families. The bZIP proteins share strong sequence and structural similarities and thus are challenging targets to bind specifically. Nevertheless, many of the designs, including examples that bind the oncoproteins c-Jun, c-Fos and c-Maf (also called JUN, FOS and MAF, respectively), were selective for their targets over all 19 other families. Collectively, the designs exhibit a wide range of interaction profiles and demonstrate that human bZIPs have only sparsely sampled the possible interaction space accessible to them. Our computational method provides a way to systematically analyse trade-offs between stability and specificity and is suitable for use with many types of structure-scoring functions; thus, it may prove broadly useful as a tool for protein design.


Current Opinion in Structural Biology | 2008

Structural specificity in coiled-coil interactions

Gevorg Grigoryan; Amy E. Keating

Coiled coils have a rich history in the field of protein design and engineering. Novel structures, such as the first seven-helix coiled coil, continue to provide surprises and insights. Large-scale datasets quantifying the influence of systematic mutations on coiled-coil stability are a valuable new asset to the area. Scoring methods based on sequence and/or structure can predict interaction preferences in coiled-coil-mediated bZIP transcription factor dimerization. Experimental and computational methods for dealing with the near-degeneracy of many coiled-coil structures appear promising for future design applications.


Nature Structural & Molecular Biology | 2002

The structure of VibH represents nonribosomal peptide synthetase condensation, cyclization and epimerization domains

Thomas A. Keating; Marshall Cg; Christopher T. Walsh; Amy E. Keating

Nonribosomal peptide synthetases (NRPSs) are large, multidomain enzymes that biosynthesize medically important natural products. We report the crystal structure of the free-standing NRPS condensation (C) domain VibH, which catalyzes amide bond formation in the synthesis of vibriobactin, a Vibrio cholerae siderophore. Despite low sequence identity, NRPS condensation enzymes are structurally related to chloramphenicol acetyltransferase (CAT) and dihydrolipoamide acyltransferases. However, although the latter enzymes are homotrimers, VibH is a monomeric pseudodimer. The VibH structure is representative of both NRPS condensation and epimerization domains, as well as the condensation-variant cyclization domains, which are all expected to be monomers. Surprisingly, despite favorable positioning in the active site, a universally conserved histidine important in CAT and in other C domains is not critical for general base catalysis in VibH.


Current Opinion in Structural Biology | 2011

Protein Binding Specificity versus Promiscuity

Gideon Schreiber; Amy E. Keating

Interactions between macromolecules in general, and between proteins in particular, are essential for any life process. Examples include transfer of information, inhibition or activation of function, molecular recognition as in the immune system, assembly of macromolecular structures and molecular machines, and more. Proteins interact with affinities ranging from millimolar to femtomolar and, because affinity determines the concentration required to obtain 50% binding, the amount of different complexes formed is very much related to local concentrations. Although the concentration of a specific binding partner is usually quite low in the cell (nanomolar to micromolar), the total concentration of other macromolecules is very high, allowing weak and non-specific interactions to play important roles. In this review we address the question of binding specificity, that is, how do some proteins maintain monogamous relations while others are clearly polygamous. We examine recent work that addresses the molecular and structural basis for specificity versus promiscuity. We show through examples how multiple solutions exist to achieve binding via similar interfaces and how protein specificity can be tuned using both positive and negative selection (specificity by demand). Binding of a protein to numerous partners can be promoted through variation in which residues are used for binding, conformational plasticity and/or post-translational modification. Natively unstructured regions represent the extreme case in which structure is obtained only upon binding. Many natively unstructured proteins serve as hubs in protein-protein interaction networks and such promiscuity can be of functional importance in biology.


Genome Biology | 2004

Predicting specificity in bZIP coiled-coil protein interactions

Jessica H. Fong; Amy E. Keating; Mona Singh

We present a method for predicting protein-protein interactions mediated by the coiled-coil motif. When tested on interactions between nearly all human and yeast bZIP proteins, our method identifies 70% of strong interactions while maintaining that 92% of predictions are correct. Furthermore, cross-validation testing shows that including the bZIP experimental data significantly improves performance. Our method can be used to predict bZIP interactions in other genomes and is a promising approach for predicting coiled-coil interactions more generally.


Journal of Molecular Biology | 2010

Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-xL

Sanjib Dutta; Stefano V. Gullá; T. Scott Chen; Emiko Fire; Robert A. Grant; Amy E. Keating

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the alpha-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques--yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis--to elucidate specificity determinants for binding to Bcl-x(L)versus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x(L) selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x(L), Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x(L)-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x(L) binders.


Proceedings of the National Academy of Sciences of the United States of America | 2001

Side-chain repacking calculations for predicting structures and stabilities of heterodimeric coiled coils.

Amy E. Keating; Vladimir N. Malashkevich; Bruce Tidor; Peter S. Kim

An important goal in biology is to predict from sequence data the high-resolution structures of proteins and the interactions that occur between them. In this paper, we describe a computational approach that can make these types of predictions for a series of coiled-coil dimers. Our method comprises a dual strategy that augments extensive conformational sampling with molecular mechanics minimization. To test the performance of the method, we designed six heterodimeric coiled coils with a range of stabilities and solved x-ray crystal structures for three of them. The stabilities and structures predicted by the calculations agree very well with experimental data: the average error in unfolding free energies is <1 kcal/mol, and nonhydrogen atoms in the predicted structures superimpose onto the experimental structures with rms deviations <0.7 Å. We have also tested the method on a series of homodimers derived from vitellogenin-binding protein. The predicted relative stabilities of the homodimers show excellent agreement with previously published experimental measurements. A critical step in our procedure is to use energy minimization to relax side-chain geometries initially selected from a rotamer library. Our results show that computational methods can predict interaction specificities that are in good agreement with experimental data.


Science | 2013

Networks of bZIP Protein-Protein Interactions Diversified Over a Billion Years of Evolution

Aaron W. Reinke; Jiyeon Baek; Orr Ashenberg; Amy E. Keating

bZIPping Through Evolution The basic region-leucine zipper (bZIP) transcription factors are found in many species and can form complexes that bind to DNA and affect transcription. Reinke et al. (p. 730) analyzed interactions for over 3000 bZIPs within and among five metazoan and two unicellular species. The results reveal differences within bZIP interactive networks that have accumulated over time and identify plasticity among interactions and changes in binding specificity that relate to specific amino acid residue changes. A comparative study of a dimeric transcription factor family looks at the evolution of protein interactions. Differences in biomolecular sequence and function underlie dramatic ranges of appearance and behavior among species. We studied the basic region-leucine zipper (bZIP) transcription factors and quantified bZIP dimerization networks for five metazoan and two single-cell species, measuring interactions in vitro for 2891 protein pairs. Metazoans have a higher proportion of heteromeric bZIP interactions and more network complexity than the single-cell species. The metazoan bZIP interactomes have broadly similar structures, but there has been extensive rewiring of connections compared to the last common ancestor, and each species network is highly distinct. Many metazoan bZIP orthologs and paralogs have strikingly different interaction specificities, and some differences arise from minor sequence changes. Our data show that a shifting landscape of biochemical functions related to signaling and gene expression contributes to species diversity.


Protein Science | 2010

Mcl-1-Bim complexes accommodate surprising point mutations via minor structural changes.

Emiko Fire; Stefano V. Gullá; Robert A. Grant; Amy E. Keating

Mcl‐1 is an antiapoptotic Bcl‐2‐family protein that protects cells against death. Structures of Mcl‐1, and of other anti‐apoptotic Bcl‐2 proteins, reveal a surface groove into which the α‐helical BH3 regions of certain proapoptotic proteins can bind. Despite high overall structural conservation, differences in this groove afford binding specificity that is important for the mechanism of Bcl‐2 family function. We report the crystal structure of human Mcl‐1 bound to a BH3 peptide derived from human Bim and the structures for three complexes that accommodate large physicochemical changes at conserved Bim sites. The mutations had surprisingly modest effects on complex stability, and the structures show that Mcl‐1 can undergo small changes to accommodate the mutant ligands. For example, a shift in a leucine side chain fills a hole left by an isoleucine‐to‐alanine mutation at the first hydrophobic buried position of Bim BH3. Larger changes are also observed, with shifting of helix α3 accommodating an isoleucine‐to‐tyrosine mutation at this same position. We surveyed the variation in available Mcl‐1 and Bcl‐xL structures and observed moderate flexibility that is likely critical for facilitating interactions of diverse BH3‐only proteins with Mcl‐1. With the antiapoptotic Bcl‐2 family members attracting significant attention as therapeutic targets, these structures contribute to our growing understanding of how specificity is achieved and can help to guide the design of novel inhibitors that target Mcl‐1.


ACS Synthetic Biology | 2012

SYNZIP protein interaction toolbox: in vitro and in vivo specifications of heterospecific coiled-coil interaction domains.

Kenneth Evan Thompson; Caleb J. Bashor; Wendell A. Lim; Amy E. Keating

The synthetic biology toolkit contains a growing number of parts for regulating transcription and translation, but very few that can be used to control protein association. Here we report characterization of 22 previously published heterospecific synthetic coiled-coil peptides called SYNZIPs. We present biophysical analysis of the oligomerization states, helix orientations, and affinities of 27 SYNZIP pairs. SYNZIP pairs were also tested for interaction in two cell-based assays. In a yeast two-hybrid screen, >85% of 253 comparable interactions were consistent with prior in vitro measurements made using coiled-coil microarrays. In a yeast-signaling assay controlled by coiled-coil mediated scaffolding, 12 SYNZIP pairs were successfully used to down-regulate the expression of a reporter gene following treatment with α-factor. Characterization of these interaction modules dramatically increases the number of available protein interaction parts for synthetic biology and should facilitate a wide range of molecular engineering applications. Summary characteristics of 27 SYNZIP peptide pairs are reported in specification sheets available in the Supporting Information and at the SYNZIP Web site [http://keatingweb.mit.edu/SYNZIP/].

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T. Scott Chen

Massachusetts Institute of Technology

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Sanjib Dutta

Massachusetts Institute of Technology

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Aaron W. Reinke

Massachusetts Institute of Technology

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Robert A. Grant

Massachusetts Institute of Technology

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Glenna W. Foight

Massachusetts Institute of Technology

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Orr Ashenberg

Massachusetts Institute of Technology

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Emiko Fire

Massachusetts Institute of Technology

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