Conformational proofreading

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Conformational proofreading or conformational selection is a general mechanism of molecular recognition systems, suggested by Yonatan Savir and Tsvi Tlusty, in which introducing an energetic barrier - such as a structural mismatch between a molecular recognizer and its target - enhances the recognition specificity and quality. [1] [2] [3] [4] [5] [6] Conformational proofreading does not require the consumption of energy and may therefore be used in any molecular recognition system. Conformational proofreading is especially useful in scenarios where the recognizer has to select the appropriate target among many similar competitors. Proteins evolve the capacity for conformational proofreading through fine-tuning their geometry, flexibility and chemical interactions with the target. [7]

Contents

Balancing correct and incorrect binding

Molecular recognition takes place in a noisy, crowded biological environment and the recognizer often has to cope with the task of selecting its target among a variety of similar competitors. For example, the ribosome has to select the correct tRNA that matches the mRNA codon among many structurally similar tRNAs. If the recognizer and its correct target match perfectly like a lock and a key, then the binding probability will be high since no deformation is required upon binding. At the same time, the recognizer might also bind to a competitor with a similar structure with high probability. Introducing an energy barrier, in particular, structural mismatch between the recognizer (lock) and the key, reduces the binding probability to the correct target but reduces even more the binding probability to a similar wrong target and thus improves the specificity. [1] [2] [3] [7] Yet, introducing too much deformation drastically reduces binding probability to the correct target. Therefore, the optimal balance between maximizing the correct binding probability and minimizing the incorrect binding probability is achieved when the recognizer is slightly off target. This suggests that conformational changes during molecular recognition processes, such as the induced fit [8] mechanism, are advantageous for enhancing the specificity of recognition. Such conformational changes may be fine-tuned by mutations that affect the mechanical response of the recognizer, also at positions far from the binding site. [7]

Conformational proofreading in homologous recombination. Top: The binding probability to homologous (correct) and non-homologous (wrong) DNA sequences decrease with the deformation energy barrier. The wrong binding probability decreases before the correct one. Bottom: As a result, the Fitness which is the difference, Fitness = Prob(Correct) - Prob(Wrong), is maximal at a non-zero deformation energy. This barrier is optimal in the sense that it significantly reduces the binding probability while keeping the correct binding probability roughly the same. Biochemical measurements of RecA-induced recombination suggest that the observed deformation is nearly optimal. Conformational proofreading in homologous recombination.jpg
Conformational proofreading in homologous recombination. Top: The binding probability to homologous (correct) and non-homologous (wrong) DNA sequences decrease with the deformation energy barrier. The wrong binding probability decreases before the correct one. Bottom: As a result, the Fitness which is the difference, Fitness = Prob(Correct) − Prob(Wrong), is maximal at a non-zero deformation energy. This barrier is optimal in the sense that it significantly reduces the binding probability while keeping the correct binding probability roughly the same. Biochemical measurements of RecA-induced recombination suggest that the observed deformation is nearly optimal.

The mechanism of conformational proofreading is utilized in the system of homologous recombination to discern between similar DNA sequences. [3] [4] Homologous recombination facilitates the exchange of genetic material between homologous DNA molecules. This crucial process requires detecting a specific homologous DNA sequence within a huge variety of heterologous sequences. The detection is mediated by RecA in E. coli, or members of its superfamily in other organisms. RecA first polymerizes along a stretch of single-stranded DNA, and then this protein-DNA filament searches for homology along double-stranded DNA. In the RecA-DNA filament, the distance between bases increases significantly with respect to the bare 3.4 Å in the double-strand (by 50% on average [9] ). This sets a significant energetic barrier on the search, since the double-stranded DNA has to stretch by the same magnitude to check for homology. By formulating the DNA recognition process as a signal detection problem, it was shown that the experimentally observed RecA-induced DNA deformation and the binding energetics are fine-tuned to ensure optimal sequence detection. The amount of deformation is such that binding to homologous DNA sequences only slightly decreases, while binding to wrong sequences decreases significantly. This is exactly the conformational proofreading mechanism.

Experimental evidence for conformational proofreading by homologous recombination

The group of C. Dekker (Delft University) directly probed the interactions involved in homology search by combining magnetic and optical tweezers. [10] They have found that homology search and recognition requires opening of the helix and can therefore be accelerated by unwinding the DNA. This is exactly the energy barrier predicted by the conformational proofreading model. The data indicate a physical picture for homology recognition in which the fidelity of the search process is governed by the distance between the DNA-binding sites. The authors conclude that their interpretation of the measurements "is akin to a conformational proofreading scheme ... where the dsDNA, and not the RecA filament, is the active, recognizing search entity. A large conformational mismatch exists between the target-bound and unbound states of the dsDNA. The target-bound state is accessed via energetically unfavorable intermediate states, as discussed above. The conformational mismatch improves the selectivity of the recognition reaction." In other words, they identified the energetic barrier and have shown that indeed the double-stranded DNA is the active participant, since it has to pass this barrier.

Use by ribosome for tRNA decoding

The ribosome is a complex molecular machine that, in order to synthesize proteins during the translation process, has to decode mRNAs by pairing their codons with matching tRNAs. Decoding is a major determinant of fitness and requires accurate and fast selection of correct tRNAs among many similar competitors. One must have in mind that most binding events are by non-matching (“non-cognate”) tRNAs and the ribosome needs to reject those as fast as possible in order to vacate the binding site. At the same time, the ribosome should keep the matching tRNAs bound long enough to allow the protein synthesis process ensue. Despite the importance of tRNA decoding, it was unclear until recently whether the modern ribosome, and in particular its large conformational changes during decoding, are the outcome of adaptation to its task as a decoder or the result of other constraints. Recent study [5] derived the energy landscape that provides optimal discrimination between competing tRNA substrates, and thereby optimal tRNA decoding. The optimal landscape is a symmetric one (see image). The study shows that the measured landscape of the prokaryotic ribosome is indeed symmetric. This model suggests that conformational changes of the ribosome and tRNA during decoding are means to obtain such an optimal tRNA decoder. The fact that both homologous recombination and tRNA decoding utilize conformational proofreading suggests that this is a generic mechanism that may be utilized broadly by molecular recognition systems.

Ribosome uses conformational proofreading for tRNA decoding. main: The curves show the free energy landscape of codon recognition as suggested by experiments. In the stages that are sensitive to codon identity, the pathways of correct (green) and wrong (red) tRNAs split. The multistage kinetics include: Initial binding and codon recognition: a complex of elongation factor (EF-Tu) and aminoacyl-tRNA binds to the ribosome. Codon is recognized by pairing with the anticodon, and by additional interaction with the "decoding center" of the ribosome. As a result, correct (cognate) tRNAs are more stable than non-cognate ones. GTP activation and hydrolysis: Codon recognition leads to global conformational changes of the ribosome and tRNA, which are different for cognate or non-cognate tRNAs and affect GTP activation and hydrolysis by EF-Tu. The conformational proofreading model explains these conformational changes as a means to enhance tRNA recognition. inset: The symmetric adapted landscape implies that the ratio of forward and backward rates is inverted between the correct and wrong energy landscapes. Ribosome uses conformational proofreading for tRNA decoding.jpg
Ribosome uses conformational proofreading for tRNA decoding. main: The curves show the free energy landscape of codon recognition as suggested by experiments. In the stages that are sensitive to codon identity, the pathways of correct (green) and wrong (red) tRNAs split. The multistage kinetics include: Initial binding and codon recognition: a complex of elongation factor (EF-Tu) and aminoacyl-tRNA binds to the ribosome. Codon is recognized by pairing with the anticodon, and by additional interaction with the "decoding center" of the ribosome. As a result, correct (cognate) tRNAs are more stable than non-cognate ones. GTP activation and hydrolysis: Codon recognition leads to global conformational changes of the ribosome and tRNA, which are different for cognate or non-cognate tRNAs and affect GTP activation and hydrolysis by EF-Tu. The conformational proofreading model explains these conformational changes as a means to enhance tRNA recognition.inset: The symmetric adapted landscape implies that the ratio of forward and backward rates is inverted between the correct and wrong energy landscapes.

In other biological systems

Human UV-damage repair

A recent study shows that conformational proofreading is used by human DNA repair mechanisms. [11] The research focused on the question of how DNA-repair proteins scan the human genome for UV-induced damage during the initial step of nucleotide excision repair (NER). Detailed single-molecule measurements revealed how the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search. The authors find that "UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2)2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude... These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection." The authors conclude from their detailed kinetic measurements that UV-DDB recognizes lesions using a conformational proofreading mechanism via multiple intermediates.

Other recognition schemes

Relation to kinetic proofreading

In the kinetic proofreading [12] [13] schema, a time delay (equivalently, an irreversible intermediate stage) is introduced during the formation of the correct or incorrect complexes. This time delay reduces the production rates of both complexes but enhances the fidelity beyond the equilibrium limit. The irreversibility of the scheme requires an energy source. The time delay in kinetic proofreading is analogous to the spatial difference in conformational proofreading. However, the conformational proofreading can be an equilibrium scheme that does not consume energy.

Related Research Articles

<span class="mw-page-title-main">Ribosome</span> Intracellular organelle consisting of RNA and protein functioning to synthesize proteins

Ribosomes are macromolecular machines, found within all cells, that perform biological protein synthesis. Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to form polypeptide chains. Ribosomes consist of two major components: the small and large ribosomal subunits. Each subunit consists of one or more ribosomal RNA (rRNA) molecules and many ribosomal proteins. The ribosomes and associated molecules are also known as the translational apparatus.

Gene knockouts are a widely used genetic engineering technique that involves the targeted removal or inactivation of a specific gene within an organism's genome. This can be done through a variety of methods, including homologous recombination, CRISPR-Cas9, and TALENs.

<span class="mw-page-title-main">Genetic recombination</span> Production of offspring with combinations of traits that differ from those found in either parent

Genetic recombination is the exchange of genetic material between different organisms which leads to production of offspring with combinations of traits that differ from those found in either parent. In eukaryotes, genetic recombination during meiosis can lead to a novel set of genetic information that can be further passed on from parents to offspring. Most recombination occurs naturally and can be classified into two types: (1) interchromosomal recombination, occurring through independent assortment of alleles whose loci are on different but homologous chromosomes ; & (2) intrachromosomal recombination, occurring through crossing over.

<span class="mw-page-title-main">Translation (biology)</span> Cellular process of protein synthesis

In molecular biology and genetics, translation is the process in which ribosomes in the cytoplasm or endoplasmic reticulum synthesize proteins after the process of transcription of DNA to RNA in the cell's nucleus. The entire process is called gene expression.

<span class="mw-page-title-main">Helicase</span> Class of enzymes to unpack an organisms genes

Helicases are a class of enzymes thought to be vital to all organisms. Their main function is to unpack an organism's genetic material. Helicases are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two hybridized nucleic acid strands, using energy from ATP hydrolysis. There are many helicases, representing the great variety of processes in which strand separation must be catalyzed. Approximately 1% of eukaryotic genes code for helicases.

<span class="mw-page-title-main">Nuclease</span> Class of enzymes

A nuclease is an enzyme capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids. Nucleases variously effect single and double stranded breaks in their target molecules. In living organisms, they are essential machinery for many aspects of DNA repair. Defects in certain nucleases can cause genetic instability or immunodeficiency. Nucleases are also extensively used in molecular cloning.

<span class="mw-page-title-main">DNA repair</span> Cellular mechanism

DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA damage, resulting in tens of thousands of individual molecular lesions per cell per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. When normal repair processes fail, and when cellular apoptosis does not occur, irreparable DNA damage may occur, including double-strand breaks and DNA crosslinkages. This can eventually lead to malignant tumors, or cancer as per the two hit hypothesis.

<span class="mw-page-title-main">Molecular lesion</span> Damage to the structure of a biological molecule

A molecular lesion or point lesion is damage to the structure of a biological molecule such as DNA, RNA, or protein. This damage may result in the reduction or absence of normal function, and in rare cases the gain of a new function. Lesions in DNA may consist of breaks or other changes in chemical structure of the helix, ultimately preventing transcription. Meanwhile, lesions in proteins consist of both broken bonds and improper folding of the amino acid chain. While many nucleic acid lesions are general across DNA and RNA, some are specific to one, such as thymine dimers being found exclusively in DNA. Several cellular repair mechanisms exist, ranging from global to specific, in order to prevent lasting damage resulting from lesions.

<span class="mw-page-title-main">Molecular recognition</span> Type of non-covalent bonding

The term molecular recognition refers to the specific interaction between two or more molecules through noncovalent bonding such as hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, π-π interactions, halogen bonding, or resonant interaction effects. In addition to these direct interactions, solvents can play a dominant indirect role in driving molecular recognition in solution. The host and guest involved in molecular recognition exhibit molecular complementarity. Exceptions are molecular containers, including e.g. nanotubes, in which portals essentially control selectivity.

<span class="mw-page-title-main">RecA</span> DNA repair protein

RecA is a 38 kilodalton protein essential for the repair and maintenance of DNA. A RecA structural and functional homolog has been found in every species in which one has been seriously sought and serves as an archetype for this class of homologous DNA repair proteins. The homologous protein is called RAD51 in eukaryotes and RadA in archaea.

<span class="mw-page-title-main">Triple-stranded DNA</span> DNA structure

Triple-stranded DNA is a DNA structure in which three oligonucleotides wind around each other and form a triple helix. In triple-stranded DNA, the third strand binds to a B-form DNA double helix by forming Hoogsteen base pairs or reversed Hoogsteen hydrogen bonds.

<span class="mw-page-title-main">Homologous recombination</span> Genetic recombination between identical or highly similar strands of genetic material

Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids.

Bacterial translation is the process by which messenger RNA is translated into proteins in bacteria.

<span class="mw-page-title-main">Holliday junction</span> Branched nucleic acid structure

A Holliday junction is a branched nucleic acid structure that contains four double-stranded arms joined. These arms may adopt one of several conformations depending on buffer salt concentrations and the sequence of nucleobases closest to the junction. The structure is named after Robin Holliday, the molecular biologist who proposed its existence in 1964.

Kinetic proofreading is a mechanism for error correction in biochemical reactions, proposed independently by John Hopfield (1974) and Jacques Ninio (1975). Kinetic proofreading allows enzymes to discriminate between two possible reaction pathways leading to correct or incorrect products with an accuracy higher than what one would predict based on the difference in the activation energy between these two pathways.

<span class="mw-page-title-main">DDB2</span> Protein-coding gene in the species Homo sapiens

DNA damage-binding protein 2 is a protein that in humans is encoded by the DDB2 gene.

<span class="mw-page-title-main">Protein synthesis inhibitor</span> Inhibitors of translation

A protein synthesis inhibitor is a compound that stops or slows the growth or proliferation of cells by disrupting the processes that lead directly to the generation of new proteins.

The term proofreading is used in genetics to refer to the error-correcting processes, first proposed by John Hopfield and Jacques Ninio, involved in DNA replication, immune system specificity, enzyme-substrate recognition among many other processes that require enhanced specificity. The proofreading mechanisms of Hopfield and Ninio are non-equilibrium active processes that consume ATP to enhance specificity of various biochemical reactions.

<span class="mw-page-title-main">Synthesis-dependent strand annealing</span>

Synthesis-dependent strand annealing (SDSA) is a major mechanism of homology-directed repair of DNA double-strand breaks (DSBs). Although many of the features of SDSA were first suggested in 1976, the double-Holliday junction model proposed in 1983 was favored by many researchers. In 1994, studies of double-strand gap repair in Drosophila were found to be incompatible with the double-Holliday junction model, leading researchers to propose a model they called synthesis-dependent strand annealing. Subsequent studies of meiotic recombination in S. cerevisiae found that non-crossover products appear earlier than double-Holliday junctions or crossover products, challenging the previous notion that both crossover and non-crossover products are produced by double-Holliday junctions and leading the authors to propose that non-crossover products are generated through SDSA.

<span class="mw-page-title-main">DNA base flipping</span> Biochemical process

DNA base flipping, or nucleotide flipping, is a mechanism in which a single nucleotide base, or nucleobase, is rotated outside the nucleic acid double helix. This occurs when a nucleic acid-processing enzyme needs access to the base to perform work on it, such as its excision for replacement with another base during DNA repair. It was first observed in 1994 using X-ray crystallography in a methyltransferase enzyme catalyzing methylation of a cytosine base in DNA. Since then, it has been shown to be used by different enzymes in many biological processes such as DNA methylation, various DNA repair mechanisms, and DNA replication. It can also occur in RNA double helices or in the DNA:RNA intermediates formed during RNA transcription.

References

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  11. ↑ Ghodke H, Wang H, Hsieh CL, Woldemeskel S, Watkins SC, Rapić-Otrin V, Van Houten B (May 2014). "Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates". Proceedings of the National Academy of Sciences of the United States of America. 111 (18): E1862–E1871. Bibcode:2014PNAS..111E1862G. doi: 10.1073/pnas.1323856111 . PMC   4020048 . PMID   24760829.
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