Kinetic Proofreading Research Articles

Discrimination of membrane antigen affinity by B cells requires dominance of kinetic proofreading over serial triggering (112.10)

Abstract B cells receptor (BCR) signaling in response to membrane-bound antigen increases with antigen affinity, a process known as affinity discrimination. However, if BCR molecules become signaling-capable immediately upon binding antigen, the decrease in serial engagement as affinity increases results in weaker signaling as affinity increases. We use computational modeling to show that a threshold time (kinetic proofreading) for antigen to stay bound to BCR before the latter becomes signaling-capable is needed to overcome the decrease in serial engagement with increasing antigen affinity, and replicate the monotonic increase in B cell signaling with affinity observed in experiments. This finding matches well with the experimentally observed timescale (~20 seconds) of antigen-mediated conformational changes to BCR that lead to Src-family kinase recruitment. The physical basis of the threshold time of antigen binding may lie in the formation timescale of BCR dimers. The latter decreases with increasing affinity, resulting in shorter threshold antigen binding times as affinity increases. Such an affinity-dependent kinetic proofreading requirement results in affinity discrimination very similar to that observed in experiments. We also develop a measure to characterize affinity discrimination in a quantitative manner. B cell affinity discrimination is critical to the process of affinity maturation, and thus our results have important implications in applications such as vaccine design.

The Ribotoxin Restrictocin Recognizes Its RNA Substrate by Selective Engagement of Active Site Residues

Restrictocin and related fungal endoribonucleases from the α-sarcin family site-specifically cleave the sarcin/ricin loop (SRL) on the ribosome to inhibit translation and ultimately trigger cell death. Previous studies showed that the SRL folds into a bulged-G motif and tetraloop, with restrictocin achieving a specificity of ∼1000-fold by recognizing both motifs only after the initial binding step. Here, we identify contacts within the protein−RNA interface and determine the extent to which each one contributes to enzyme specificity by examining the effect of protein mutations on the cleavage of the SRL substrate compared to a variety of other RNA substrates. As with other biomolecular interfaces, only a subset of contacts contributes to specificity. One contact of this subset is critical, with the H49A mutation resulting in quantitative loss of specificity. Maximum catalytic activity occurs when both motifs of the SRL are present, with the major contribution involving the bulged-G motif recognized by three lysine residues located adjacent to the active site: K110, K111, and K113. Our findings support a kinetic proofreading mechanism in which the active site residues H49 and, to a lesser extent, Y47 make greater catalytic contributions to SRL cleavage than to suboptimal substrates. This systematic and quantitative analysis begins to elucidate the principles governing RNA recognition by a site-specific endonuclease and may thus serve as a mechanistic model for investigating other RNA modifying enzymes.

Remote Toehold: A Mechanism for Flexible Control of DNA Hybridization Kinetics

Hybridization of DNA strands can be used to build molecular devices, and control of the kinetics of DNA hybridization is a crucial element in the design and construction of functional and autonomous devices. Toehold-mediated strand displacement has proved to be a powerful mechanism that allows programmable control of DNA hybridization. So far, attempts to control hybridization kinetics have mainly focused on the length and binding strength of toehold sequences. Here we show that insertion of a spacer between the toehold and displacement domains provides additional control: modulation of the nature and length of the spacer can be used to control strand-displacement rates over at least 3 orders of magnitude. We apply this mechanism to operate displacement reactions in potentially useful kinetic regimes: the kinetic proofreading and concentration-robust regimes.

Distribution of dwell times of a ribosome: effects of infidelity, kinetic proofreading and ribosome crowding

Ribosome is a molecular machine that polymerizes a protein where the sequence of the amino acid residues, the monomers of the protein, is dictated by the sequence of codons (triplets of nucleotides) on a messenger RNA (mRNA) that serves as the template. The ribosome is a molecular motor that utilizes the template mRNA strand also as the track. Thus, in each step the ribosome moves forward by one codon and, simultaneously, elongates the protein by one amino acid. We present a theoretical model that captures most of the main steps in the mechanochemical cycle of a ribosome. The stochastic movement of the ribosome consists of an alternating sequence of pause and translocation; the sum of the durations of a pause and the following translocation is the time of dwell of the ribosome at the corresponding codon. We derive the analytical expression for the distribution of the dwell times of a ribosome in our model. Wherever experimental data are available, our theoretical predictions are consistent with those results. We suggest appropriate experiments to test the new predictions of our model, particularly the effects of the quality control mechanism of the ribosome and that of their crowding on the mRNA track.

Proofreading and spellchecking: A two-tier strategy for pre-mRNA splicing quality control

Multi-tier strategies exist in many biochemical processes to ensure a maximal fidelity of the reactions. In this review, we focus on the two-tier quality control strategy that ensures the quality of the products of the pre-mRNA splicing reactions catalyzed by the spliceosome. The first step in the quality control process relies on kinetic proofreading mechanisms that are internal to the spliceosome and that are performed by ATP-dependent RNA helicases. The second quality control step, spellchecking, involves recognition of unspliced pre-mRNAs or aberrantly spliced mRNAs that have escaped the first proofreading mechanisms, and subsequent degradation of these molecules by degradative enzymes in the nucleus or in the cytoplasm. This two-tier quality control strategy highlights a need for high fidelity and a requirement for degradative activities that eliminate defective molecules. The presence of multiple quality control activities during splicing underscores the importance of this process in the expression of genetic information.

The Role of Initiator tRNAi met in Fidelity of Initiation of Protein Synthesis

The proper arrangement of amino acids in a protein determines its proper function, which is vital for the cellular metabolism. This indicates that the process of peptide bond formation requires high fidelity. One of the most important processes for this fidelity is kinetic proofreading. As biochemical experiments suggest that kinetic proofreading plays a major role in ensuring the fidelity of protein synthesis, it is not certain whether or not a misacylated tRNA would be corrected by kinetic proofreading during the peptide bond formation. Using 2-layered ONIOM (QM/MM) computational calculations, we studied the behavior of misacylated tRNAs and compared the results with these for cognate aminoacyl-tRNAs during the process of peptide bond formation to investigate the effect of nonnative amino acids on tRNAs. The difference between the behavior of initiator tRNAi met compared to the one for the elongator tRNAs indicates that only the initiator tRNAi met specifies the amino acid side chain.

Kinetic proofreading and the search for nonself-peptides

The T cells scan the surface of antigen-presenting cells with their T cell receptors (TCR) in order to find and respond to specific peptide-major histocompatibility complexes (pMHC). Since mainly self-peptides are expressed on antigen-presenting cells, the T cells must utilize sensitive mechanisms in order to quickly discriminate between self and nonself-peptides. A range of different models have been proposed to account for this process. Due to the experimental inconsistency of how T cells respond to altered peptides it has been difficult to validate the competing models. Recent models, based on the kinetic proofreading model, propose that a short life-time of the TCR/pMHC complexes may be compensated by fast rebinding of the individual molecules. Hence, both the on- and off-rate involved in the interaction between pMHCs and TCRs will determine the fate of the T cell discrimination. I here briefly review some of the proposed models on T cell discrimination and scanning, and discuss the significance of determining self-peptide kinetics to validate the different models.

A proposal for kinetic proof reading by ISWI family chromatin remodeling motors

ATP-dependent chromatin remodeling motors play fundamental roles in nuclear processes by regulating access to DNA. Yet compared to other cellular motors less is known about how these motors couple the energy of ATP to alter their substrates. Here we use recent studies on a key chromatin remodeling motor from the ISWI class, human ACF and its yeast counterpart, ISW2, to propose a model for how these motors use ATP to read structural cues presented by nucleosomal substrates. Substantial earlier work has shown that ACF activity is strongly regulated by the length of the DNA flanking a nucleosome as well as by the histone H4 tail. Recent bulk and single-molecule studies of human ACF suggest that this complex functions as a dimeric motor. These studies, together with studies of yeast ISW2 imply that at least two types of ATP hydrolysis events accompany each cycle of nucleosome movement. We propose that ISWI motors may employ a kinetic proof reading type of mechanism to favor action on nucleosomes that are poised to be in condensed chromatin while inhibiting action on nucleosomes that are in fully active or fully condensed chromatin.

A detailed mathematical model predicts that serial engagement of IgE-Fc epsilon RI complexes can enhance Syk activation in mast cells.

The term serial engagement was introduced to describe the ability of a single peptide, bound to a MHC molecule, to sequentially interact with TCRs within the contact region between a T cell and an APC. In addition to ligands on surfaces, soluble multivalent ligands can serially engage cell surface receptors with sites on the ligand, binding and dissociating from receptors many times before all ligand sites become free and the ligand leaves the surface. To evaluate the role of serial engagement in Syk activation, we use a detailed mathematical model of the initial signaling cascade that is triggered when FcepsilonRI is aggregated on mast cells by multivalent Ags. Although serial engagement is not required for mast cell signaling, it can influence the recruitment of Syk to the receptor and subsequent Syk phosphorylation. Simulating the response of mast cells to ligands that serially engage receptors at different rates shows that increasing the rate of serial engagement by increasing the rate of dissociation of the ligand-receptor bond decreases Syk phosphorylation. Increasing serial engagement by increasing the rate at which receptors are cross-linked (for example by increasing the forward rate constant for cross-linking or increasing the valence of the ligand) increases Syk phosphorylation. When serial engagement enhances Syk phosphorylation, it does so by partially reversing the effects of kinetic proofreading. Serial engagement rapidly returns receptors that have dissociated from aggregates to new aggregates before the receptors have fully returned to their basal state.

Fast on-rates allow short dwell time ligands to activate T cells.

Two contrasting theories have emerged that attempt to describe T-cell ligand potency, one based on the t(1/2) of the interaction and the other based on the equilibrium affinity (K(D)). Here, we have identified and studied an extensive set of T-cell receptor (TCR)-peptide-MHC (pMHC) interactions for CD4(+) cells that have differential K(D)s and kinetics of binding. Our data indicate that ligands with a short t(1/2) can be highly stimulatory if they have fast on-rates. Simple models suggest these fast kinetic ligands are stimulatory because the pMHCs bind and rebind the same TCR several times. Rebinding occurs when the TCR-pMHC on-rate outcompetes TCR-pMHC diffusion within the cell membrane, creating an aggregate t(1/2) (t(a)) that can be significantly longer than a single TCR-pMHC encounter. Accounting for t(a), ligand potency is K(D)-based when ligands have fast on-rates (k(on)) and t(1/2)-dependent when they have slow k(on). Thus, TCR-pMHC k(on) allow high-affinity short t(1/2) ligands to follow a kinetic proofreading model.

Constraining errors in splice site choice

While even single nucleotide errors in pre‐mRNA splicing can lead to catastrophic consequences, our understanding of fidelity mechanisms in splicing is poor. Genetics studies have established the importance of fidelity mechanisms in splicing and implicated 3 of the 8 spliceosomal DExD/H box ATPases in competing with splicing to enable kinetic proofreading. However, due to a paucity of in vitro assays, it has remained unclear how specificity is established by kinetic proofreading and how rejected substrates are discarded. We have established in vitro that each chemical step in splicing is proofread by a DEAH box ATPase – 5′ splice site cleavage is proofread by Prp16 and exon ligation is proofread by Prp22. In a kinetic proofreading mechanism, specificity can be achieved if the rate of the ATPase and/or the rate of the proofread step varies with the authenticity of the substrate. We have found evidence that both Prp16 and Prp22 enhance specificity by discriminating against slowly splicing substrates. Both Prp16 and Prp22 reject substrates reversibly, necessitating an independent discard activity that we attribute to the DEAH box ATPase Prp43, which normally functions to discard a genuine intron after excision from the precursor. These findings establish that both chemical steps in splicing are proofread by a common ATP‐dependent framework and support a general role for DExD/H box ATPases in fidelity. Support: ACS, NIH.

Computational modeling of B cell signaling shows B cell affinity discrimination requires kinetic proofreading (84.2)

Abstract B cells signaling in response to antigen is proportional to antigen affinity, a process known as affinity discrimination. B cells can acquire antigen in membrane-bound form on the surface of antigen-presenting cells (APCs), with signaling being initiated within a few seconds of contact. During the early stages of contact, B cell receptors (BCRs) on protrusions of the B cell surface bind to antigen on the APC surface and form micro-clusters of 10-100 BCR-antigen complexes. We use a Monte Carlo computational model to show that B cell affinity discrimination at the level of micro-clusters requires a threshold antigen binding time, similar to kinetic proofreading. We find that if BCR molecules become signaling-capable immediately upon binding antigen, there is a loss in serial engagement as BCR-antigen affinity increases, due to the increased bond lifetime of high-affinity bonds. This results in an unnatural decrease in signaling strength with increasing affinity. A threshold antigen binding time favors high affinity BCR-antigen bonds, as these long-lived bonds can better fulfill the threshold time requirement than low-affinity bonds. A threshold antigen binding time of ~10 seconds results in monotonically increasing signaling with affinity, as seen in B cell activation experiments. This time matches well with the experimentally observed time (~20 seconds) of lipid raft-mediated conformational changes in BCR signaling domains that lead to association with Syk.

Dependence of T Cell Antigen Recognition on T Cell Receptor-Peptide MHC Confinement Time

SummaryT cell receptor (TCR) binding to diverse peptide-major histocompatibility complex (pMHC) ligands results in various degrees of T cell activation. Here we analyze which binding properties of the TCR-pMHC interaction are responsible for this variation in pMHC activation potency. We have analyzed activation of the 1G4 cytotoxic T lymphocyte clone by cognate pMHC variants and performed thorough correlation analysis of T cell activation with 1G4 TCR-pMHC binding properties measured in solution. We found that both the on rate (kon) and off rate (koff) contribute to activation potency. Based on our results, we propose a model in which rapid TCR rebinding to the same pMHC after chemical dissociation increases the effective half-life or “confinement time” of a TCR-pMHC interaction. This confinement time model clarifies the role of kon in T cell activation and reconciles apparently contradictory reports on the role of TCR-pMHC binding kinetics and affinity in T cell activation.

B Cell Affinity Discrimination Requires Kinetic Proofreading

B cells signaling in response to antigen is proportional to antigen affinity, a process known as affinity discrimination. Recent research suggests that B cells can acquire antigen in membrane-bound form on the surface of antigen-presenting cells (APCs), with signaling being initiated within a few seconds of B cell/APC contact. During the earliest stages of B cell/APC contact, B cell receptors (BCRs) on protrusions of the B cell surface bind to antigen on the APC surface and form micro-clusters of 10-100 BCR/Antigen complexes. In this study, we use computational modeling to show that B cell affinity discrimination at the level of BCR-antigen micro-clusters requires a threshold antigen binding time, in a manner similar to kinetic proofreading. We find that if BCR molecules become signaling-capable immediately upon binding antigen, the loss in serial engagement due to the increase in bond lifetime as affinity increases results in a considerable decrease in signaling with increasing affinity. Adding a threshold antigen binding time for BCR to become signaling-capable favors high affinity BCR-antigen bonds, as these long-lived bonds can better fulfill the threshold time requirement than low-affinity bonds. A threshold antigen binding time of ∼10 seconds for BCR to become signaling-capable results in monotonically increasing signaling with affinity, replicating the affinity discrimination pattern observed in B cell activation experiments. This time matches well (within order of magnitude) with the experimentally observed time (∼ 20 seconds) required for the BCR signaling domains to undergo antigen and lipid raft-mediated conformational changes that lead to association with Syk.

The simplicity of completion time distributions for common complex biochemical processes

Biochemical processes typically involve huge numbers of individual reversible steps, each with its own dynamical rate constants. For example, kinetic proofreading processes rely upon numerous sequential reactions in order to guarantee the precise construction of specific macromolecules. In this work, we study the transient properties of such systems and fully characterize their first passage (completion) time distributions. In particular, we provide explicit expressions for the mean and the variance of the completion time for a kinetic proofreading process and computational analyses for more complicated biochemical systems. We find that, for a wide range of parameters, as the system size grows, the completion time behavior simplifies: it becomes either deterministic or exponentially distributed, with a very narrow transition between the two regimes. In both regimes, the dynamical complexity of the full system is trivial compared to its apparent structural complexity. Similar simplicity is likely to arise in the dynamics of many complex multistep biochemical processes. In particular, these findings suggest not only that one may not be able to understand individual elementary reactions from macroscopic observations, but also that such an understanding may be unnecessary.

Specificity and completion time distributions of biochemical processes

In order to produce specific complex structures from a large set of similar biochemical building blocks, many biochemical systems require high sensitivity to small molecular differences. The first and most common model used to explain this high specificity is kinetic proofreading, which has been extended to a variety of systems from detection of DNA mismatch to cell signaling processes. While the specification properties of kinetic proofreading models are well known and were studied in various contexts, very little is known about their temporal behavior. In this work, we study the dynamical properties of discrete stochastic two-branch kinetic proofreading schemes. Using the Laplace transform of the corresponding chemical master equation, we obtain an analytical solution for the completion time distribution. In particular we provide expressions for the specificity as well as the mean and variance of the process completion times. We also show that, for a wide range of parameters, a process distinguishing between two different products can be reduced to a much simpler three-point process. Our results allow for the systematic study of the interplay between specificity and completion times, as well as testing the validity of the kinetic proofreading model in biological systems.

A Role for Rebinding in Rapid and Reliable T Cell Responses to Antigen

Experimental work has shown that T cells of the immune system rapidly and specifically respond to antigenic molecules presented on the surface of antigen-presenting-cells and are able to discriminate between potential stimuli based on the kinetic parameters of the T cell receptor-antigen bond. These antigenic molecules are presented among thousands of chemically similar endogenous peptides, raising the question of how T cells can reliably make a decision to respond to certain antigens but not others within minutes of encountering an antigen presenting cell. In this theoretical study, we investigate the role of localized rebinding between a T cell receptor and an antigen. We show that by allowing the signaling state of individual receptors to persist during brief unbinding events, T cells are able to discriminate antigens based on both their unbinding and rebinding rates. We demonstrate that T cell receptor coreceptors, but not receptor clustering, are important in promoting localized rebinding, and show that requiring rebinding for productive signaling reduces signals from a high concentration of endogenous pMHC. In developing our main results, we use a relatively simple model based on kinetic proofreading. However, we additionally show that all our results are recapitulated when we use a detailed T cell receptor signaling model. We discuss our results in the context of existing models and recent experimental work and propose new experiments to test our findings.

Elucidation of T cell signalling models

A potential mechanism that allows T cells to reliably discriminate pMHC ligands involves an interplay between kinetic proofreading, negative feedback and a destruction of this negative feedback. We analyse a detailed model of these mechanisms which involves the TCR, SHP1 and ERK. We discover that the behaviour of pSHP1 negative feedback is of primary importance, and particularly the influence of a kinetic proofreading base negative feedback state on pSHP1 dynamics. The CD8 co-receptor is shown to benefit from a kinetic proofreading locking mechanism and is able to overcome pSHP1 negative influences to sensitise a T cell.

Mechanical Forces in T Cell Triggering

T cell is one of the main player of mammalian immune response. It ensures antigen recognition at the surface of antigen presenting cells (APCs) in a complex highly sensitive and specific process where encounter of T cell receptor with agonist peptide associated with major histocompatibility complex triggers T cell activation. Despite its central role, the mechanism of TCR triggering is still unclear. Several models involving receptor oligomerisation, kinetic proofreading, serial triggering are currently under debate.We present here a work aimed to explore experimentally the role of mechanical cues in T cell activation.We will show the first results of mechanical engagement of TCR in Jurkat cell line using magnetic particles and related cell response as reported by intracellular Ca2+ transient.We will discuss then how these results could provide a mechanistic solution to the TCR triggering puzzle.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Visualization of the Hybrid State of tRNA Binding Promoted by Spontaneous Ratcheting of the Ribosome

A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunit is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69, and H38 that is caused by the ratcheting of the small subunit.