Timothy Travers

Modeling the Assembly of the Multiple Domains of α-actinin-4 and Its Role in Actin Cross-linking

The assembly of proteins into multidomain complexes is critical for their function. In eukaryotic nonmuscle cells, regulation of the homodimeric actin cross-linking protein α-actinin-4 (ACTN4) during cell migration involves signaling receptors with intrinsic tyrosine kinase activity, yet the underlying molecular mechanisms are poorly understood. As a first step to address the latter, we validate here an atomic model for the ACTN4 end region, which corresponds to a ternary complex between the N-terminal actin-binding domain (ABD) and an adjacent helical neck region of one monomer, and the C-terminal calmodulin-like domain of the opposite antiparallel monomer. Mutagenesis experiments designed to disrupt this ternary complex confirm that its formation reduces binding to F-actin. Molecular dynamics simulations show that the phosphomimic mutation Y265E increases actin binding by breaking several interactions that tether the two calponin homology domains into a closed ABD conformation. Simulations also show a disorder-to-order transition in the double phosphomimic mutant Y4E/Y31E of the 45-residue ACTN4 N-terminal region, which can inhibit actin binding by latching both calponin homology domains more tightly. Collectively, these studies provide a starting point for understanding the role of external cues in regulating ACTN4, with different phenotypes resulting from changes in the multidomain assembly of the protein.

The carboxyl tail of alpha-actinin-4 regulates its susceptibility to m-calpain and thus functions in cell migration and spreading

Alpha-actinin-4 links the cytoskeleton to sites of adhesion and has been shown to be modulated to enable cell migration. Such focal adhesions must be labile to accomplish migration, with this detachment occurring at least in part via m-calpain activation (Glading et al., 2001, 2002; Xie et al., 1998). In this study, we report that alpha-actinin-4 is initially cleaved by m-calpain between tyrosine 13 and glycine. Removal of the first 13 amino acids does not affect alpha-actinin-4 binding to actin filaments and its localization within fibroblasts but drives cell migration with less persistence. Binding of phosphoinositides PI(4,5)P2, PI(3,4,5)P3 and PI(3,4)P2 to alpha-actinin-4, as well as binding of alpha-actinin-4 to actin filaments all inhibit m-calpain cleavage of ACTN4 between tyrosine 13 and glycine 14. Interestingly, the carboxyl terminus of alpha-actinin-4 including its calcium binding motifs, is inhibitory for a secondary cleavage of alpha-actinin-4 between lysine 283 and valine 284. The minimal length of inhibitory domain is mapped to the last 11 amino acids of alpha-actinin-4. The C-terminal tail of alpha-actinin-4 is essential for maintaining its normal actin binding activity and localization within cytoplasm and also its colocalization with actin in the lamellipodia of locomoting fibroblasts. Live cell imaging reveals that the 1-890 fragment fails to rescue neither the basal or growth factor-stimulated migration nor the revert the spread area of fibroblasts to the level of NR6WT. These findings suggest that the C-terminal tail of alpha-actinin-4 is essential for its function in cell migration and adhesion to substratum.

Tandem phosphorylation within an intrinsically disordered region regulates ACTN4 function

Phosphorylation of a tyrosine in the disordered N-terminal region of ACTN4 functions as a switch exposing a second site for phosphorylation. Flipping the phosphorylation switch How does phosphorylation of a protein at one site regulate phosphorylation of a second site? Travers et al. identified distinct roles for tandem phosphorylation sites in an intrinsically disordered region of α-actinin-4 (ACTN4). Molecular dynamics simulations, validated by experimental observations, indicated that phosphorylation on Tyr4 increased the accessibility of Tyr31 and thus phosphorylation of this site, which reduced ACTN4 binding to actin. Thus, the first site functioned as a switch that enabled phosphorylation at the second site, which controlled binding to actin. Tandem-site phosphorylation may be a mechanism by which spatiotemporal regulation of protein function evolved. The kinase for the first site may only be present or active at restricted locations or times, whereas the kinase for the second site may be constitutively active or ubiquitous, may have a loose consensus motif, or may be the same as the kinase for the first site, but has different kinetics for the second site. Phosphorylated residues occur preferentially in the intrinsically disordered regions of eukaryotic proteins. In the disordered amino-terminal region of human α-actinin-4 (ACTN4), Tyr4 and Tyr31 are phosphorylated in cells stimulated with epidermal growth factor (EGF), and a mutant with phosphorylation-mimicking mutations of both tyrosines exhibits reduced interaction with actin in vitro. Cleavage of ACTN4 by m-calpain, a protease that in motile cells is predominantly activated at the rear, removes the Tyr4 site. We found that introducing a phosphomimetic mutation at only Tyr31 was sufficient to inhibit the interaction with actin in vitro. However, molecular dynamics simulations predicted that Tyr31 is mostly buried and that phosphorylation of Tyr4 would increase the solvent exposure and thus kinase accessibility of Tyr31. In fibroblast cells, EGF stimulation increased tyrosine phosphorylation of a mutant form of ACTN4 with a phosphorylation-mimicking residue at Tyr4, whereas a truncated mutant representing the product of m-calpain cleavage exhibited EGF-stimulated tyrosine phosphorylation at a background amount similar to that observed for a double phosphomimetic mutant of Tyr4 and Tyr31. We also found that inhibition of the receptor tyrosine kinases of the TAM family, such as AXL, blocked EGF-stimulated tyrosine phosphorylation of ACTN4. Mathematical modeling predicted that the kinetics of phosphorylation at Tyr31 can be dictated by the kinase affinity for Tyr4. This study suggests that tandem-site phosphorylation within intrinsically disordered regions provides a mechanism for a site to function as a switch to reveal a nearby function-regulating site.

Anti-citrullinated heat shock protein 90 antibodies identified in bronchoalveolar lavage fluid are a marker of lung-specific immune responses☆

Previous work has demonstrated a correlation between serum anti-citrullinated HSP90 antibodies and rheumatoid arthritis-associated interstitial lung disease (RA-ILD). To further investigate this potential pathogenic relationship, we used ELISA-based techniques to assess anti-citrullinated HSP90 antibody profiles in bronchoalveolar lavage fluid (BALF) of patients with different stages of RA-ILD. 9/21 RA-derived BALF specimens demonstrated IgG and/or IgA antibodies targeting citrullinated HSP90 proteins/peptides, highlighting disease specific responses (with a predilection for RA-ILD) that did not occur in IPF patients (0/5) or healthy control subjects (0/5). Comparison of antibody profiles between BALF and matching serum specimens revealed various recognition patterns favoring predominant production of anti-citrullinated HSP90 antibodies within the lung microenvironment-further supporting the connection between this antibody specificity and parenchymal lung disease. Equally important, qualitative as well as quantitative differences in anti-citrullinated HSP90 profiles between BALF and serum indicate that the lung plays a direct role in shaping the immune repertoire of RA/RA-ILD.

FixingTIM: interactive exploration of sequence and structural data to identify functional mutations in protein families

BackgroundKnowledge of the 3D structure and functionality of proteins can lead to insight into the associated cellular processes, speed up the creation of pharmaceutical products, and develop drugs that are more effective in combating disease.MethodsWe present the design and implementation of a visual mining and analysis tool to help identify protein mutations across a family of structural models and to help discover the effect of these mutations on protein function. We integrate 3D structure and sequence information in a common visual interface; multiple linked views and a computational backbone allow comparison at the molecular and atomic levels, while a novel trend-image visual abstraction allows for the sorting and mining of large collections of sequences and of their residues.ResultsWe evaluate our approach on the triosephosphate isomerase (TIM) family structural models and sequence data and show that our tool provides an effective, scalable way to navigate a family of proteins, as well as a means to inspect the structure and sequence of individual proteins.ConclusionsThe TIM application shows that our tool can assist in the navigation of families of proteins, as well as in the exploration of individual protein structures. In conjunction with domain expert knowledge, this interactive tool can help provide biophysical insight into why specific mutations affect function and potentially suggest additional modifications to the protein that could be used to rescue functionality.

Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain

Activation of RAF kinase involves the association of its RAS-binding domain (RBD) and cysteine-rich domain (CRD) with membrane-anchored RAS. However, the overall architecture of the RAS/RBD/CRD ternary complex and the orientations of its constituent domains at the membrane remain unclear. Here, we have combined all-atom and coarse-grained molecular dynamics (MD) simulations with experimental data to construct and validate a model of membrane-anchored CRD, and used this as a basis to explore models of membrane-anchored RAS/RBD/CRD complex. First, simulations of the CRD revealed that it anchors to the membrane via insertion of its two hydrophobic loops, which is consistent with our NMR measurements of CRD bound to nanodiscs. Simulations of the CRD in the context of membrane-anchored RAS/RBD then show how CRD association with either RAS or RBD could play an unexpected role in guiding the membrane orientations of RAS/RBD. This finding has implications for the formation of RAS-RAS dimers, as different membrane orientations of RAS expose distinct putative dimerization interfaces.

Extensive Citrullination Promotes Immunogenicity of HSP90 through Protein Unfolding and Exposure of Cryptic Epitopes

Post-translational protein modifications such as citrullination have been linked to the breach of immune tolerance and clinical autoimmunity. Previous studies from our laboratory support this concept, demonstrating that autoantibodies targeting citrullinated isoforms of heat shock protein 90 (HSP90) are associated with rheumatoid arthritis complicated by interstitial lung disease. To further explore the relationship between citrullination and structural determinants of HSP90 immunogenicity, we employed a combination of ELISA-based epitope profiling, computational modeling, and mass-spectrometric sequencing of peptidylarginine deiminase (PAD)-modified protein. Remarkably, ELISAs involving selected citrullinated HSP90β/α peptides identified a key epitope corresponding to an internal Arg residue (R502 [HSP90β]/R510 [HSP90α]) that is normally buried within the crystal structure of native/unmodified HSP90. In vitro time/dose-response experiments reveal an ordered pattern of PAD-mediated deimination events culminating in citrullination of R502/R510. Conventional as well as scaled molecular dynamics simulations further demonstrate that citrullination of selected Arg residues leads to progressive disruption of HSP90 tertiary structure, promoting exposure of R502/R510 to PAD modification and subsequent autoantibody binding. Consistent with this process, ELISAs incorporating variably deiminated HSP90 as substrate Ag indicate a direct relationship between the degree of citrullination and the level of ex vivo Ab recognition. Overall, these data support a novel structural paradigm whereby citrullination-induced shifts in protein structure generate cryptic epitopes capable of bypassing B cell tolerance in the appropriate genetic context.