Native-State Interconversion of a Metamorphic Protein Requires Global Unfolding

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Biochemistry (Impact Factor: 3.19). 08/2011; 50(33):7077-9. DOI: 10.1021/bi200750k
Source: PubMed

ABSTRACT Lymphotactin (Ltn) is a unique chemokine that under physiological solution conditions displays large-scale structural heterogeneity, defining a new category of "metamorphic proteins". Previous Ltn studies have indicated that each form is required for proper function, but the mechanism of interconversion remains unknown. Here we have investigated the temperature dependence of kinetic rates associated with interconversion and unfolding by stopped-flow fluorescence to determine transition-state free energies. Comparisons of derived thermodynamic parameters revealed striking similarities between interconversion and protein unfolding. We conclude that Ltn native-state rearrangement proceeds by way of a large-scale unfolding process rather than a unique intermediate structure.

Download full-text


Available from: Brian F Volkman, Aug 08, 2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: The mammalian chemokine family is segregated into four families - CC, CXC, CX3C, and XC - based on the arrangement of cysteines and the corresponding disulfides. Sequencing of the Danio rerio (zebrafish) genome has identified more than double the amount of human chemokines with the absence of the CX3C family and the presence of a new family, CX. The only other family with a single cysteine in the N-terminal region is the XC family. Human lymphotactin (XCL1) has two interconverting structures due to dynamic changes that occur in the protein. Similar to an experiment with XCL1 that identified the two structural forms, we probed for multiple forms of zCXL1 using heparin affinity. The results suggest only a single form of CXL1 is present. We used sulfur-SAD phasing to determine the three-dimensional structure CXL1. Zebrafish CXL1 (zCXL1) has three disulfides that appear to be important for a stable structure. One disulfide is common to all chemokines except those that belong to the XC family, another is similar to a subset of CC chemokines containing three disulfides, but the third disulfide is unique to the CX family. We analyzed the electrostatic potential of the zCXL1 structure and identified the likely heparin-binding site for glycosaminoglycans (GAGs). zCXL1 has a similar sequence identity with human CCL5 and CXCL12, but the structure is more related to CCL5. Our structural analysis supports the phylogenetic and genomic studies on the evolution of the CXL family. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
    Proteins Structure Function and Bioinformatics 05/2014; 82(5). DOI:10.1002/prot.24380 · 2.92 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Protein conformational switches alter their shape upon receiving an input signal, such as ligand binding, chemical modification, or change in environment. The apparent simplicity of this transformation--which can be carried out by a molecule as small as a thousand atoms or so--belies its critical importance to the life of the cell as well as its capacity for engineering by humans. In the realm of molecular switches, proteins are unique because they are capable of performing a variety of biological functions. Switchable proteins are therefore of high interest to the fields of biology, biotechnology, and medicine. These molecules are beginning to be exploited as the core machinery behind a new generation of biosensors, functionally regulated enzymes, and "smart" biomaterials that react to their surroundings. As inspirations for these designs, researchers continue to analyze existing examples of allosteric proteins. Recent years have also witnessed the development of new methodologies for introducing conformational change into proteins that previously had none. Herein we review examples of both natural and engineered protein switches in the context of four basic modes of conformational change: rigid-body domain movement, limited structural rearrangement, global fold switching, and folding-unfolding. Our purpose is to highlight examples that can potentially serve as platforms for the design of custom switches. Accordingly, we focus on inducible conformational changes that are substantial enough to produce a functional response (e.g., in a second protein to which it is fused), yet are relatively simple, structurally well-characterized, and amenable to protein engineering efforts.
    Chemistry - A European Journal 10/2012; 18(26):7984-99. DOI:10.1002/chem.201200348 · 5.70 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The involvement of chemokines and chemokine receptors in a great variety of pathological indications underscores their utility as therapeutic targets. In general, chemokine-mediated migration and signaling requires three distinct interactions: self-association, glycosaminoglycan (GAG) binding, and activation of G protein-coupled receptors (GPCRs). Solution-state nuclear magnetic resonance (NMR) spectroscopy has long been used to determine the apo structure of chemokines and monitor complex formation; however, it has never contributed directly to drug discovery efforts that are traditionally focused on the previously inaccessible chemokine receptors. Our lab recently demonstrated that NMR structures can be successfully utilized to direct drug discovery against chemokines. The ease of collecting chemokine structural data coupled with the increased efficiency of structure-based drug discovery campaigns makes chemokine-directed therapies particularly attractive. In addition, recent advances in sample preparation, spectrometer hardware, and pulse program development are allowing researchers to examine interactions with previously inaccessible partners - including full-length chemokine receptors. These developments will facilitate exploration of novel ways to modulate chemokine activity using structure-guided drug discovery.
    Drug Discovery Today Technologies 12/2012; 9(4):e293-e299. DOI:10.1016/j.ddtec.2012.04.003