Yihong Chen

A minimal sequence code for switching protein structure and function

We present here a structural and mechanistic description of how a protein changes its fold and function, mutation by mutation. Our approach was to create 2 proteins that (i) are stably folded into 2 different folds, (ii) have 2 different functions, and (iii) are very similar in sequence. In this simplified sequence space we explore the mutational path from one fold to another. We show that an IgG-binding, 4β+α fold can be transformed into an albumin-binding, 3-α fold via a mutational pathway in which neither function nor native structure is completely lost. The stabilities of all mutants along the pathway are evaluated, key high-resolution structures are determined by NMR, and an explanation of the switching mechanism is provided. We show that the conformational switch from 4β+α to 3-α structure can occur via a single amino acid substitution. On one side of the switch point, the 4β+α fold is >90% populated (pH 7.2, 20 °C). A single mutation switches the conformation to the 3-α fold, which is >90% populated (pH 7.2, 20 °C). We further show that a bifunctional protein exists at the switch point with affinity for both IgG and albumin.

The design and characterization of two proteins with 88% sequence identity but different structure and function

To identify a simplified code for conformational switching, we have redesigned two natural proteins to have 88% sequence identity but different tertiary structures: a 3-α helix fold and an α/β fold. We describe the design of these homologous heteromorphic proteins, their structural properties as determined by NMR, their conformational stabilities, and their affinities for their respective ligands: IgG and serum albumin. Each of these proteins is completely folded at 25°C, is monomeric, and retains the native binding activity. The complete binding epitope for both ligands is encoded within each of the proteins. The IgG-binding epitope is functional only in the α/β fold, and the albumin-binding epitope is functional only in the 3-α fold. These results demonstrate that two monomeric folds and two different functions can be encoded with only 12% of the amino acids in a protein (7 of 56). The fact that 49 aa in these proteins are compatible with both folds shows that the essential information determining a fold can be highly concentrated in a few amino acids and that a very limited subset of interactions in the protein can tip the balance from one monomer fold to another. This delicate balance helps explain why protein structure prediction is so challenging. Furthermore, because a few mutations can result in both new conformation and new function, the evolution of new folds driven by natural selection for alternative functions may be much more probable than previously recognized.

NMR structures of two designed proteins with high sequence identity but different fold and function

How protein sequence codes for 3D structure remains a fundamental question in biology. One approach to understanding the folding code is to design a pair of proteins with maximal sequence identity but retaining different folds. Therefore, the nonidentities must be responsible for determining which fold topology prevails and constitute a fold-specific folding code. We recently designed two proteins, GA88 and GB88, with 88% sequence identity but different folds and functions [Alexander et al. (2007) Proc Natl Acad Sci USA 104:11963–11968]. Here, we describe the detailed 3D structures of these proteins determined in solution by NMR spectroscopy. Despite a large number of mutations taking the sequence identity level from 16 to 88%, GA88 and GB88 maintain their distinct wild-type 3-α and α/β folds, respectively. To our knowledge, the 3D-structure determination of two monomeric proteins with such high sequence identity but different fold topology is unprecedented. The geometries of the seven nonidentical residues (of 56 total) provide insights into the structural basis for switching between 3-α and α/β conformations. Further mutation of a subset of these nonidentities, guided by the GA88 and GB88 structures, leads to proteins with even higher levels of sequence identity (95%) and different folds. Thus, conformational switching to an alternative monomeric fold of comparable stability can be effected with just a handful of mutations in a small protein. This result has implications for understanding not only the folding code but also the evolution of new folds.

Phosphorylation-Induced Conformational Ensemble Switching in an Intrinsically Disordered Cancer/Testis Antigen

Background: PAGE4, an intrinsically disordered protein up-regulated in prostate cancer, binds to c-Jun and potentiates its transactivation. Results: The effects of phosphorylation on PAGE4 conformation, dynamics, and c-Jun binding were determined by NMR. Conclusion: Phosphorylation induces a more compact conformational ensemble, restricting access to the c-Jun binding site. Significance: This study may help to explain how phosphorylation of PAGE4 alters its binding to c-Jun. Prostate-associated gene 4 (PAGE4) is an intrinsically disordered cancer/testis antigen that is up-regulated in the fetal and diseased human prostate. Knocking down PAGE4 expression results in cell death, whereas its overexpression leads to a growth advantage of prostate cancer cells (Zeng, Y., He, Y., Yang, F., Mooney, S. M., Getzenberg, R. H., Orban, J., and Kulkarni, P. (2011) The cancer/testis antigen prostate-associated gene 4 (PAGE4) is a highly intrinsically disordered protein. J. Biol. Chem. 286, 13985–13994). Phosphorylation of PAGE4 at Thr-51 is critical for potentiating c-Jun transactivation, an important factor in controlling cell growth, apoptosis, and stress response. Using NMR spectroscopy, we show that the PAGE4 polypeptide chain has local and long-range conformational preferences that are perturbed by site-specific phosphorylation at Thr-51. The population of transient turn-like structures increases upon phosphorylation in an ∼20-residue acidic region centered on Thr-51. This central region therefore becomes more compact and more negatively charged, with increasing intramolecular contacts to basic sequence motifs near the N and C termini. Although flexibility is decreased in the central region of phospho-PAGE4, the polypeptide chain remains highly dynamic overall. PAGE4 utilizes a transient helical structure adjacent to the central acidic region to bind c-Jun with low affinity in vitro. The binding interaction is attenuated by phosphorylation at Thr-51, most likely because of masking the effects of the more compact phosphorylated state. Therefore, phosphorylation of PAGE4 leads to conformational shifts in the dynamic ensemble, with large functional consequences. The changes in the structural ensemble induced by posttranslational modifications are similar conceptually to the conformational switching events seen in some marginally stable (“metamorphic”) folded proteins in response to mutation or environmental triggers.

A nuclear magnetic resonance based approach to accurate functional annotation of putative enzymes in the methanogen Methanosarcina acetivorans

BackgroundCorrect annotation of function is essential if one is to take full advantage of the vast amounts of genomic sequence data. The accuracy of sequence-based functional annotations is often variable, particularly if the sequence homology to a known function is low. Indeed recent work has shown that even proteins with very high sequence identity can have different folds and functions, and therefore caution is needed in assigning functions by sequence homology in the absence of experimental validation. Experimental methods are therefore needed to efficiently evaluate annotations in a way that complements current high throughput technologies. Here, we describe the use of nuclear magnetic resonance (NMR)-based ligand screening as a tool for testing functional assignments of putative enzymes that may be of variable reliability.ResultsThe target genes for this study are putative enzymes from the methanogenic archaeon Methanosarcina acetivorans (MA) that have been selected after manual genome re-annotation and demonstrate detectable in vivo expression at the level of the transcriptome. The experimental approach begins with heterologous E. coli expression and purification of individual MA gene products. An NMR-based ligand screen of the purified protein then identifies possible substrates or products from a library of candidate compounds chosen from the putative pathway and other related pathways. These data are used to determine if the current sequence-based annotation is likely to be correct. For a number of case studies, additional experiments (such as in vivo genetic complementation) were performed to determine function so that the reliability of the NMR screen could be independently assessed.ConclusionsIn all examples studied, the NMR screen was indicative of whether the functional annotation was correct. Thus, the case studies described demonstrate that NMR-based ligand screening is an effective and rapid tool for confirming or negating the annotated gene function of putative enzymes. In particular, no protein-specific assay needs to be developed, which makes the approach broadly applicable for validating putative functions using an automated pipeline strategy.

Identification of the Docking Site for CD3 on the T Cell Receptor β Chain by Solution NMR

Background: Understanding T cell signaling requires knowing the structure of the TCR-CD3 complex. Results: Solution NMR was used to identify the docking site for CD3 ectodomains on the TCR β chain. Conclusion: The docking site (∼400 Å2) comprises ∼10 Cβ residues at the base of the TCR. Significance: CD3 is located opposite to the peptide-MHC binding site of the TCR. The T cell receptor (TCR)-CD3 complex is composed of a genetically diverse αβ TCR heterodimer associated noncovalently with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates peptide-MHC recognition, whereas the CD3 molecules transduce activation signals to the T cell. Although much is known about downstream T cell signaling pathways, the mechanism whereby TCR engagement by peptide-MHC initiates signaling is poorly understood. A key to solving this problem is defining the spatial organization of the TCR-CD3 complex and the interactions between its subunits. We have applied solution NMR methods to identify the docking site for CD3 on the β chain of a human autoimmune TCR. We demonstrate a low affinity but highly specific interaction between the extracellular domains of CD3 and the TCR constant β (Cβ) domain that requires both CD3ϵγ and CD3ϵδ subunits. The mainly hydrophilic docking site, comprising 9–11 solvent-accessible Cβ residues, is relatively small (∼400 Å2), consistent with the weak interaction between TCR and CD3 extracellular domains, and devoid of glycosylation sites. The docking site is centered on the αA and αB helices of Cβ, which are located at the base of the TCR. This positions CD3ϵγ and CD3ϵδ between the TCR and the T cell membrane, permitting us to distinguish among several possible models of TCR-CD3 association. We further correlate structural results from NMR with mutational data on TCR-CD3 interactions from cell-based assays.

Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity

Significance The onset of androgen resistance is a major impediment in treating prostate cancer (PCa). However, the underlying molecular mechanisms are not fully understood. Here, we integrate multiple biophysical approaches that report conformational preferences of the intrinsically disordered protein (IDP) Prostate-Associated Gene 4 (PAGE4) with human cancer biology and nonlinear dynamics. Based on our biophysical, biochemical, and cellular data, a mathematical model is presented that suggests a mechanism by which phosphorylation-induced conformational changes in PAGE4 can switch from an androgen-dependent phenotype to an androgen-independent phenotype in PCa cells. The study underscores how IDPs that engage in multiple “promiscuous” interactions due to their conformational dynamics when overexpressed or aberrantly expressed can stochastically orchestrate phenotypic heterogeneity in PCa. Intrinsically disordered proteins (IDPs) that lack a unique 3D structure and comprise a large fraction of the human proteome play important roles in numerous cellular functions. Prostate-Associated Gene 4 (PAGE4) is an IDP that acts as a potentiator of the Activator Protein-1 (AP-1) transcription factor. Homeodomain-Interacting Protein Kinase 1 (HIPK1) phosphorylates PAGE4 at S9 and T51, but only T51 is critical for its activity. Here, we identify a second kinase, CDC-Like Kinase 2 (CLK2), which acts on PAGE4 and hyperphosphorylates it at multiple S/T residues, including S9 and T51. We demonstrate that HIPK1 is expressed in both androgen-dependent and androgen-independent prostate cancer (PCa) cells, whereas CLK2 and PAGE4 are expressed only in androgen-dependent cells. Cell-based studies indicate that PAGE4 interaction with the two kinases leads to opposing functions. HIPK1-phosphorylated PAGE4 (HIPK1-PAGE4) potentiates c-Jun, whereas CLK2-phosphorylated PAGE4 (CLK2-PAGE4) attenuates c-Jun activity. Consistent with the cellular data, biophysical measurements (small-angle X-ray scattering, single-molecule fluorescence resonance energy transfer, and NMR) indicate that HIPK1-PAGE4 exhibits a relatively compact conformational ensemble that binds AP-1, whereas CLK2-PAGE4 is more expanded and resembles a random coil with diminished affinity for AP-1. Taken together, the results suggest that the phosphorylation-induced conformational dynamics of PAGE4 may play a role in modulating changes between PCa cell phenotypes. A mathematical model based on our experimental data demonstrates how differential phosphorylation of PAGE4 can lead to transitions between androgen-dependent and androgen-independent phenotypes by altering the AP-1/androgen receptor regulatory circuit in PCa cells.

PAGE4 and Conformational Switching: Insights from Molecular Dynamics Simulations and Implications for Prostate Cancer

Prostate-associated gene 4 (PAGE4) is an intrinsically disordered protein implicated in prostate cancer. Thestress-response kinase homeodomain-interacting protein kinase 1 (HIPK1) phosphorylates two residues in PAGE4, serine 9 and threonine 51. Phosphorylation of these two residues facilitates the interaction of PAGE4 with activator protein-1 (AP-1) transcription factor complex to potentiate AP-1s activity. In contrast, hyperphosphorylation of PAGE4 by CDC-like kinase 2 (CLK2) attenuates this interaction with AP-1. Small-angleX-ray scattering and single-molecule fluorescence resonance energy transfer measurements have shown that PAGE4 expands upon hyperphosphorylation and that this expansion is localized to its N-terminal half. To understand the interactions underlying this structural transition, we performed molecular dynamics simulations using Atomistic AWSEM, a multi-scale molecular model that combines atomistic and coarse-grained simulation approaches. Our simulations show that electrostatic interactions drive transient formation of an N-terminal loop, the destabilization of which accounts for the dramatic change in size upon hyperphosphorylation. Phosphorylation also changes the preference of secondary structure formation of the PAGE4 ensemble, which leads to a transition between states that display different degrees of disorder. Finally, we construct a mechanism-based mathematical model that allows us to capture the interactions ofdifferent phosphoforms of PAGE4 with AP-1 and its downstream target, the androgen receptor (AR)-a key therapeutic target in prostate cancer. Our model predicts intracellular oscillatory dynamics of HIPK1-PAGE4, CLK2-PAGE4, and AR activity, indicating phenotypic heterogeneity in an isogenic cell population. Thus, conformational switching of PAGE4 may potentially affect the efficiency of therapeutically targeting AR activity.

Solution NMR structure of a sheddase inhibitor prodomain from the malarial parasite Plasmodium falciparum

Plasmodium subtilisin 2 (Sub2) is a multidomain protein that plays an important role in malaria infection. Here, we describe the solution NMR structure of a conserved region of the inhibitory prodomain of Sub2 from Plasmodium falciparum, termed prosub2. Despite the absence of any detectable sequence homology, the protozoan prosub2 has structural similarity to bacterial and mammalian subtilisin‐like prodomains. Comparison with the three‐dimensional structures of these other prodomains suggests a likely binding interface with the catalytic domain of Sub2 and provides insights into the locations of primary and secondary processing sites in Plasmodium prodomains. Proteins 2012;.

Structural metamorphism and polymorphism in proteins on the brink of thermodynamic stability: Continuum of Order/Disorder Transitions

The classical view of the structure–function paradigm advanced by Anfinsen in the 1960s is that a proteins function is inextricably linked to its three‐dimensional structure and is encrypted in its amino acid sequence. However, it is now known that a significant fraction of the proteome consists of intrinsically disordered proteins (IDPs). These proteins populate a polymorphic ensemble of conformations rather than a unique structure but are still capable of performing biological functions. At the boundary, between well‐ordered and inherently disordered states are proteins that are on the brink of stability, either weakly stable ordered systems or disordered but on the verge of being stable. In such marginal states, even relatively minor changes can significantly alter the energy landscape, leading to large‐scale conformational remodeling. Some proteins on the edge of stability are metamorphic, with the capacity to switch from one fold topology to another in response to an environmental trigger (e.g., pH, temperature/salt, redox). Many IDPs, on the other hand, are marginally unstable such that small perturbations (e.g., phosphorylation, ligands) tip the balance over to a range of ordered, partially ordered, or even more disordered states. In general, the structural transitions described by metamorphic fold switches and polymorphic IDPs possess a number of common features including low or diminished stability, large‐scale conformational changes, critical disordered regions, latent or attenuated binding sites, and expansion of function. We suggest that these transitions are, therefore, conceptually and mechanistically analogous, representing adjacent regions in the continuum of order/disorder transitions.