Native-State Interconversion of a Metamorphic Protein Requires Global Unfolding

Tyler, Robert C.; Murray, Nathan J.; Peterson, Francis C.; Volkman, Brian F.

doi:10.1021/bi200750k

Cited by 46 publications

(102 citation statements)

References 16 publications

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“…7 This restructuring is accompanied by changes in the overall distribution of electrostatic charges on the surface of XCL1 (Figure 1A–D). XCL1 mon contains 15 basic and six acidic residues (Figure 1E), resulting in a theoretical isoelectric point (pI) of 10.6.…”

Section: Resultsmentioning

confidence: 99%

“…4,6 The XCL1 dim conformation is an unrelated β -sandwich dimer that is responsible for binding to GAGs. 5 Both native state structures can be accessed through a dynamic equilibrium unfolding process, 7 designated as metamorphic interconversion.…”

mentioning

confidence: 99%

“…5,7,8 For example, Tyler et al identified interactions that allow the folding equilibrium of XCL1 mon and XCL1 dim to respond to perturbations in ionic strength. 8 XCL1 mon is destabilized by the electrostatic repulsion existing between Arg23 and Arg43 because of their proximity within the secondary structure.…”

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confidence: 99%

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Examination of Glycosaminoglycan Binding Sites on the XCL1 Dimer

et al. 2016

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Known for its distinct metamorphic behavior, XCL1 interconverts between a canonical chemokine folded monomer (XCL1mon) that interacts with the receptor, XCR1, and a unique dimer (XCL1dim) that interacts with glycosaminoglycans and inhibits HIV-1 activity. This study presents the first detailed analysis of the GAG binding properties of XCL1dim. Basic residues within a conformationally selective dimeric variant of XCL1 (W55D) were mutated and analyzed for their effects on heparin binding. Mutation of Arg23 and Arg43 greatly diminished the level of heparin binding in both heparin Sepharose chromatography and surface plasmon resonance assays. To assess the contributions of different GAG structures to XCL1 binding, we developed a solution fluorescence polarization assay and correlated affinity with the length and level of sulfation of heparan sulfate oligosaccharides. It was recently demonstrated that the XCL1 GAG binding form, XCL1dim, is responsible for preventing HIV-1 infection through interactions with gp120. This study defines a GAG binding surface on XCL1dim that includes residues that are important for HIV-1 inhibition.

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Section: Resultsmentioning

confidence: 99%

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Examination of Glycosaminoglycan Binding Sites on the XCL1 Dimer

et al. 2016

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“…4 Interconversion between these two folds involves a complete restructuring of the core residues. 5 Since the development of soft ionization techniques such as electrospray ionization (ESI) and nano-ESI (n-ESI), mass spectrometry (MS) has become a valuable tool for structural analysis of proteins and protein complexes, and methods have been established that can preserve solution topologies. 6−9 Furthermore, theoretical studies have suggested that major secondary structural features of proteins can be preserved during transfer into the gas phase under ESI conditions.…”

Section: ■ Introductionmentioning

confidence: 99%

Dissecting the Dynamic Conformations of the Metamorphic Protein Lymphotactin

Harvey¹,

Porrini

Konijnenberg³

et al. 2014

J. Phys. Chem. B

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A mass spectrometer provides an ideal laboratory to probe the structure and stability of isolated protein ions. Interrogation of each discrete mass/charge-separated species enables the determination of the intrinsic stability of a protein fold, gaining snapshots of unfolding pathways. In solution, the metamorphic protein lymphotactin (Ltn) exists in equilibrium between two distinct conformations, a monomeric (Ltn10) and a dimeric (Ltn40) fold. Here, we use electron capture dissociation (ECD) and drift tube ion mobility-mass spectrometry (DT IM-MS) to analyze both forms and use molecular dynamics (MD) to consider how the solution fold alters in a solvent-free environment. DT IM-MS reveals significant conformational flexibility for the monomer, while the dimer appears more conformationally restricted. These findings are supported by MD calculations, which reveal how salt bridges stabilize the conformers in vacuo. Following ECD experiments, a distinctive fragmentation pattern is obtained for both the monomer and dimer. Monomer fragmentation becomes more pronounced with increasing charge state especially in the disordered regions and C-terminal α-helix in the solution fold. Lower levels of fragmentation are seen in the β-sheet regions and in regions that contain salt bridges, identified by MD simulations. The lowest charge state of the dimer for which we obtain ECD data ([D+9H](9+)) exhibits extensive fragmentation with no relationship to the solution fold and has a smaller collision cross section (CCS) than charge states 10-13+, suggesting a "collapsed" encounter complex. Other charge states of the dimer, as for the monomer, are resistant to fragmentation in regions of β-sheets in the solution fold. This study provides evidence for preservation and loss of global fold and secondary structural elements, providing a tantalizing glimpse into the power of the emerging field of native top-down mass spectrometry.

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“…Therefore, it is likely that the interconversion requires the complete unfolding and refolding, rather than a well-structured intermediate; this mechanistic hypothesis is consistent with the kinetic analyses of hLtn10/ hLtn40 interconversion and unfolding of the two species. 17 The hLtn system represents a remarkable example for how environmental properties, such as salt and temperature modulate protein stability through the interplay of electrostatics and hydrophobic interactions. 18−20 Prior to the solution of the NMR structure for hLtn40, 12 our molecular dynamics (MD) studies 21,22 focused on probing changes in structural stability of hLtn10 at the scale of tens of nanoseconds when temperature and salt concentration was varied separately.…”

Section: ■ Introductionmentioning

confidence: 99%

Interplay of Electrostatics and Hydrophobic Effects in the Metamorphic Protein Human Lymphotactin

2015

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The human lymphotactin (hLtn) is a protein that features two native states both of which are physiologically relevant: it is a monomer (hLtn10) at 10 °C with 200 mM salt and a dimer (hLtn40) at 40 °C and without salt. Here we focus on the networks of electrostatic and hydrophobic interactions that display substantial changes upon the conversion from hLtn10 to hLtn40 since they are expected to modulate the relative stability of the two folds. In addition to the Arg 23-Arg 43 interaction discussed in previous work, we find several other like-charge pairs that are likely important to the stability of hLtn10. Free energy perturbation calculations are carried out to explicitly evaluate the contribution of the Arg 23-Arg 43 interaction to the hLtn10 stability. hLtn40 features a larger number of salt bridges, and a set of hydrophobic residues undergo major changes in the solvent accessible surface area between hLtn10 and hLtn40, pointing to their importance to the relative stability of the two folds. We also discuss the use of explicit and implicit solvent simulations for characterizing the conformational ensembles under different solution conditions.

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Native-State Interconversion of a Metamorphic Protein Requires Global Unfolding

Cited by 46 publications

References 16 publications

Examination of Glycosaminoglycan Binding Sites on the XCL1 Dimer

Examination of Glycosaminoglycan Binding Sites on the XCL1 Dimer

Dissecting the Dynamic Conformations of the Metamorphic Protein Lymphotactin

Interplay of Electrostatics and Hydrophobic Effects in the Metamorphic Protein Human Lymphotactin

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