The Dimerization of an α/β-Knotted Protein Is Essential for Structure and Function

Summaryα/β-Knotted proteins are an extraordinary example of biological self-assembly; they contain a deep topological trefoil knot formed by the backbone polypeptide chain. Evidence suggests that all are dimeric and function as methyltransferases, and the deep knot forms part of the active site. We investigated the significance of the dimeric structure of the α/β-knot protein, YibK, from Haemophilus influenzae by the design and engineering of monomeric versions of the protein, followed by examination of their structural, functional, stability, and kinetic folding properties. Monomeric forms of YibK display similar characteristics to an intermediate species populated during the formation of the wild-type dimer. However, a notable loss in structure involving disruption to the active site, rendering it incapable of cofactor binding, is observed in monomeric YibK. Thus, dimerization is vital for preservation of the native structure and, therefore, activity of the protein

Abbreviation MTase, methyltransferase

The protein-folding problem continues to be a major challenge for structural, molecular and computational biologists. The past two decades have seen the folding pathways of many proteins characterized in detail using experimental and computational approaches. Current theories suggest that proteins can collapse, rearrange, form intermediates and even swap parts of their structure in order to reach their native conformation Why are protein knots so unexpected? In its simplest form, the protein-folding problem can be broken down into two parts: first, how a given amino acid sequence specifies the final functional structure of a protein and, second, how a protein reaches this native state from an initially unfolded (or denatured) chain. Answers to these questions will have practical consequences in medicine, drug development and bio-and nanotechnolog

Evolution of RNA-Protein Interactions: Non-Specific Binding Led to RNA Splicing Activity of Fungal Mitochondrial Tyrosyl-tRNA Synthetases

<div><p>The <i>Neurospora crassa</i> mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) evolved a new function as a group I intron splicing factor by acquiring the ability to bind group I intron RNAs and stabilize their catalytically active RNA structure. Previous studies showed: (i) CYT-18 binds group I introns by using both its N-terminal catalytic domain and flexibly attached C-terminal anticodon-binding domain (CTD); and (ii) the catalytic domain binds group I introns specifically via multiple structural adaptations that occurred during or after the divergence of Peziomycotina and Saccharomycotina. However, the function of the CTD and how it contributed to the evolution of splicing activity have been unclear. Here, small angle X-ray scattering analysis of CYT-18 shows that both CTDs of the homodimeric protein extend outward from the catalytic domain, but move inward to bind opposite ends of a group I intron RNA. Biochemical assays show that the isolated CTD of CYT-18 binds RNAs non-specifically, possibly contributing to its interaction with the structurally different ends of the intron RNA. Finally, we find that the yeast mtTyrRS, which diverged from Pezizomycotina fungal mtTyrRSs prior to the evolution of splicing activity, binds group I intron and other RNAs non-specifically via its CTD, but lacks further adaptations needed for group I intron splicing. Our results suggest a scenario of constructive neutral (i.e., pre-adaptive) evolution in which an initial non-specific interaction between the CTD of an ancestral fungal mtTyrRS and a self-splicing group I intron was “fixed” by an intron RNA mutation that resulted in protein-dependent splicing. Once fixed, this interaction could be elaborated by further adaptive mutations in both the catalytic domain and CTD that enabled specific binding of group I introns. Our results highlight a role for non-specific RNA binding in the evolution of RNA-binding proteins.</p></div

Equilibrium binding of CYT-18 NTDs and the CTD to various RNAs.

<p>Binding assays of CYT-18 NTDs (blue) or CTD (red) to the (A–C) <i>N. crassa</i> mt LSU (Nc mt LSU), <i>N. crassa ND1</i>m (Nc <i>ND1</i>m), and Twort ribozyme group I intron RNAs, respectively; (D) <i>L. lactis</i> Ll.LtrB group II intron RNA; and (E) poly(U)₃₀. For the binding assays, ³²P-labeled RNAs were incubated with increasing concentrations of protein at 25°C for 30 min, a time verified to be sufficient to reach equilibrium (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002028#s4" target="_blank">Materials and Methods</a>). The plots show the fraction of RNA bound to a nitrocellulose filter as a function of protein concentration with the CYT-18 NTDs-group I intron binding data fit to hyperbolic curves and the CYT-18 NTDs-Ll.LtrB and all CYT-18 CTD-RNA binding data fit to sigmoidal curves. Dissociation constant (<i>K</i>_d) or <i>K</i>_1/2 values and Hill coefficient (n) are shown in (A–E) and are the mean for three experiments with the error bars indicating the standard deviation. Equilibrium-binding assays for the same proteins and RNAs at 37°C are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002028#pbio.1002028.s005" target="_blank">Figure S4</a>.</p

Biochemical analysis of CYT-18/Sc mtTyrRS chimeric proteins.

<p>Chimera 1 consists of the CYT-18 NTDs fused to the Sc mtTyrRS linker region and CTD, while chimera 2 consists of the CYT-18 NTDs and linker region (including Ins 3) fused to Sc CTD. Tyrosyl-adenylation, aminoacylation, and RNA-splicing assays were done at 30°C, as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002028#s4" target="_blank">Materials and Methods</a>. (A) Tyrosyl-adenylation activity is displayed as a bar graph showing the mean for three experiments, with the error bars indicating the standard deviation. (B) Aminoacylation assay showing the formation of [³H]-Tyr-tRNA^Tyr over a 60-min time course (black open circles, wild-type CYT-18; purple open squares, chimera 1; pink open diamonds, chimera 2; black closed circles, no protein). (C, D) End-point splicing assays of ³²P-labeled precursor RNAs (200 nM) containing the Nc mt LSU and <i>ND1</i>m group I introns, respectively, with 100 nM protein and 1 mM GTP for 60 min at 30°C in splicing reaction medium containing 25 or 100 mM KCl. Additional end-point splicing assays with ³²P-labeled precursor RNA at these protein and RNA concentrations at 25°C and 37°C are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002028#pbio.1002028.s008" target="_blank">Figure S7A and S7B</a>. (E, F) Splicing time courses of ³²P-labeled precursor RNA containing the <i>ND1</i>m intron (200 nM) with 100 nM protein and 1 nM GTP at 30°C in splicing reaction medium containing 25 mM or 100 mM KCl, respectively. The plots show disappearance of precursor RNA as a function of time (black open circles, wild-type CYT-18; purple open squares, chimera 1; pink open diamonds, chimera 2). (G) End-point splicing assays with unlabeled precursor RNA containing 200 nM Nc mt LSU intron and [α-³²P]GTP (500 nM; 3,000 Ci mmol⁻¹) with 500 nM protein. Reactions were incubated for 60 min at 30°C and 37°C. Darker exposures of the same gels are shown below. Minor labeled products in the CYT-18 lanes, including one co-migrating with precursor RNA and others migrating above precursor RNA (not shown), appear in time-course experiments after the 5′-labeled intron products and likely reflect secondary reactions catalyzed by group I intron RNAs (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002028#pbio.1002028-Inoue1" target="_blank">[87]</a>). The left panel shows splicing reactions for the same concentrations of wild-type CYT-18 protein and ³²P-labeled precursor RNA at 30°C run in parallel as a control. Abbreviations: E1-E2, ligated exons; E1-I, 5′ exon+intron; I, excised intron; I-E2, intron+3′ exon; P, precursor RNA.</p

χ and NSD values for protein <i>ab initio</i> models.

a<p>The discrepancy value (χ) describes the fit of the <i>ab initio</i> model to the experimental scattering curve. χ values close to 1 indicate a good fit between the models and SAXS data.</p>b<p>The NSD describes the similarity between different <i>ab initio</i> models produced by DAMMIN or GASBOR. A low value for the NSD (∼1) indicates the models are similar to each other.</p><p>χ and NSD values for protein <i>ab initio</i> models.</p

SAXS analysis of the CYT-18 protein constructs.

<p>(A) Scattering curves for the CYT-18 NTDs, CTD, and CYT-18*. The plots show the log of the scattering intensity (<i>I</i>, arbitrary units [a.u.]) as a function of momentum transfer (<i>q</i> = 4πsin(θ)/λ) and are displaced along the <i>y</i>-axis for visualization. The top and middle curves for CYT-18 NTDs and CTD show the SAXS data (black) overlaid with the expected scattering profiles calculated by CRYSOL from the CYT-18 NTDs crystal structure (blue, PDB:1Y42; χ = 1.9) or a CYT-18 CTD homology model based on the <i>A. nidulans</i> mtTyrRS CTD NMR structure (red, PDB:2KTL; χ = 2.8), respectively. The bottom curve for CYT-18* shows the SAXS data (black) overlaid with the calculated scattering profile for the CORAL model of this protein (gray; χ = 1.8). (B) Normalized pair distance distribution functions (<i>P(r)</i>) calculated from the scattering profiles by AUTOGNOM for CYT-18 CTD (red), CYT-18 NTDs (blue), and CYT-18* (gray). The <i>P(r)</i> curves for the CYT-18 NTDs and CTD are single peaks with a short tail, consistent with an elongated protein shape. (C–E) <i>Ab initio</i> models built by DAMMIN with the fit of the SAXS envelope to the corresponding high-resolution structure (CYT-18 NTDs), homology model (CYT-18 CTD), or CORAL model (CYT-18*) superimposed by SUPCOMB shown below the model <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002028#pbio.1002028-MBKozin1" target="_blank">[85]</a>. The χ values shown in parentheses in the figure indicate the fit of the <i>ab initio</i> models to the scattering data.</p

SAXS analysis and rigid-body modeling of CYT-18 proteins bound to the Twort ribozyme.

<p>(A, B) Scattering profiles of the CYT-18 NTDs and CYT-18* bound to the Twort ribozyme displaced along the logarithm axis for visualization. (A) shows CRYSOL fits of the SAXS curves (black) to theoretical scattering curves calculated from the CYT-18 NTDs+Twort co-crystal structure (blue; χ = 4.4) and CYT-18+Twort RNA model structure based on biochemical data <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002028#pbio.1002028-Paukstelis4" target="_blank">[37]</a> (gray; χ = 8.8). The CYT-18 NTDs+Twort RNA co-crystal structure and CYT-18+Twort RNA biochemical model structure are shown alongside the plots together with the <i>R</i>_g values calculated from the SAXS data and the structures. (B) shows fits of the SAXS curves (black) to the CORAL models of the CYT-18 NTDs+Twort (blue; χ = 2.2) and CYT-18*+Twort (gray; χ = 3.2). The CORAL models are shown alongside the plots together with the radii of gyration (<i>R</i>_g) calculated from the SAXS data and the models. (C, D) CORAL and biochemical models of the CYT-18+Twort RNA complex, respectively. Three views of the models are shown with the CYT-18 NTDs colored blue and the two CTDs of the homodimer colored red. The unmodeled flexible linkers in the biochemical model are shown as dashed lines. The Twort group I intron RNA is depicted in gray cartoon representation with the P4–P6 domain in orange and the P3–P9 domain in cyan. Parts of intron RNA regions P2, P4–P5, P6–P6a, P8, and P9 are near and potentially interact with the CTDs.</p