蛋白质结构域重组构建新功能酶
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摘要
酶是具有催化功能的蛋白质，其催化的多样性已使其在生物产业中得到广泛应用。应用自然界酶分子进化原理，构建具有实际应用所需的高催化效率、高稳定性的酶是蛋白质工程研究的重要目标。随着人们对酶的三级结构了解的增加，越来越多的合理设计方法，比如定点突变和结构域重组，被用来改变酶的性质和功能。通过合理设计改造的酶，不仅在工业和医药等方面起着重要作用，也加深了人们对于蛋白质结构-功能关系的认识。结构域重组是自然界中蛋白质进化的一个重要途径，也是人工设计和改造酶蛋白的有效策略。天然存在的与多种功能和性质相关的蛋白质结构域为人们构建新型酶蛋白提供了重组素材。传统的结构域重组方法通过重组同源蛋白质的结构域，已经成功地构建出了许多嵌合酶。它们或具有新的催化活力，或改变了底物特异性，或在稳定性上获得了提高，等等。一般来讲，重组亲本的同源性越低，嵌合酶获得新功能的可能性越大。重组低同源性的亲本蛋白质的困难在于，来源于不同亲本的结构域在重组过程中往往会破坏结构域界面上的相互作用，导致无活力的嵌合酶。因此，蛋白质工程的合理设计需要加深对蛋白质进化机制的理解，以及有效地构建嵌合蛋白质的新策略。本文通过两种途径重组了低同源性亲本的结构域，并成功获得了有活力的嵌合酶：（1）传统结构域重组结合结构域界面优化的方法；（2）关键基序指导的结构域重组（Key Motif Directed Recombination, KMDR）。
（一）传统结构域重组结合结构域界面优化获得嵌合酶：来自超嗜热矿泉古菌Aeropyrum pernix K1的嗜热酯酶apAPH和来自嗜热古菌Archaeoglobusfulgidus的嗜热酯酶AFEST具有相似的催化结构域，而它们的底物结合结构域差异较大。apAPH偏好水解中等长度脂肪酸链的酯底物，如pNPC8；AFEST偏好水解短脂肪酸链的酯底物，如pNPC4。为了研究apAPH和AFEST的结构功能关系，我们交换了这两个酶的底物结合结构域，并对新形成的结构域界面进行优化获得了两个有活力的嵌合酶PAR和AAM7。对这两个嵌合酶的性质表征结果表明它们都继承了亲本蛋白质的嗜热性和热稳定性。酶催化动力学结果表明，PAR和AAM7对酯酶底物的链长特异性分别与向它们提供底物结合结构域的亲本一致。我们的实验结果表明：一，底物结合结构域对于酶的底物特异性具有决定性作用；二，低同源性亲本蛋白质进行结构域重组时，对新形成的结构域界面进行优化是获得正确折叠的嵌合酶以及提高嵌合酶稳定性的重要保证。
（二）关键基序指导的结构域重组（KMDR）：同一超家族中的蛋白质成员之间具有相似的折叠结构，但在一级序列和生物功能上可能有很大的差异。在某些成员中可以检测到的蛋白质序列基序和结构基序的存在表明这些成员可能是从一个或多个共同的祖先进化来的。通过对α/β水解酶超家族成员的序列和结构的比对，我们发现了关键基序的存在。关键基序是指存在于保守的结构基序中的保守的序列基序。关键基序代表了蛋白质超家族中成员的相似的局部结构环境。我们设想可以在关键基序区域选择重组位点，利用关键基序的保守性和它与邻近结构的相互作用，重组不同亲本的结构域构建新的嵌合酶。我们提出了一个新的结构域重组的方法—关键基序指导的结构域重组（KMDR）。利用KMDR，以来自于α/β水解酶超家族的三个成员—嗜热酯酶AFEST，嗜热酯酶apAPH和中温脂肪酶Lip1（来源于褶皱假丝酵母Candida rugosa）—为亲本，设计并构建了嗜热的嵌合酶LAf和LAp。对嵌合酶的性质测定结果表明，与我们的预期一致，嵌合酶获得了嗜热亲本AFEST和apAPH的嗜热性和热稳定性，同时也保持了中温脂肪酶亲本Lip1对长链底物的偏好性。我们通过定点突变对嵌合酶LAp进行优化，获得了活力提高4.6倍的突变体。
蛋白质超家族成员中对所需功能的相关结构域进行重组是产生功能跃迁以及扩大合成蛋白质种类的有效途径。作为结构域重组的新方法，KMDR在处于刚性的结构域内部的关键基序区域进行结构域重组，而不是传统重组方法选择的结构域间的柔性连接区域。它有效地重组低同源亲本蛋白质，构建新功能嵌合蛋白质，这对于基础酶学研究以及构建在医药、化工等产业用酶都具有重要意义。我们的研究工作不仅发展了蛋白质超家族中低同源亲本结构域重组的方法，而且首次提出了关键基序在自然界蛋白质进化过程中可能具有重要作用。
Constructing enzymes with desired properties is a major goal for proteinengineers. Recombination is an important mechanism for natural evolution of proteins,and presents an effective strategy for exploring protein sequence space. Diversefunctional domains in proteins provide a resource for designing novel biocatalysts.The recombination of functional protein modules, such as domains or subdomains,from diverse homologous enzymes by conventional domain recombination has led tochimeric enzymes with novel activities, altered substrate specificities, and improvedstability. Recombining proteins with less sequence similarity would offer betteropportunities for creating novel proteins. The challenge has been that the domainsfrom parental proteins with less sequence identity often have structurallynon-compatible interfaces when they are recombined, resulting in inactive chimeras.Thus, further advances in rational protein engineering require a profoundunderstanding of how proteins evolve and new strategies for the efficient generationof chimeras. In this work, we constructed chimeras through two different strategies:traditional domain recombination combining with domain interface refinement; andKey Motif Directed Recombination.
AFEST is a carboxylesterase from Archaeoglobus fulgidus, hydrolyzesp-nitrophenyl esters (pNP) with short acyl chain lengths, and shows the highestactivity towards pNPC4(para-nitrophenyl-butyrate). apAPH is an acylpeptidehydrolase from Aeropyrum pernix K1(apAPH) and shows a promiscuous esteraseactivity with a preference for middle chain length substrates, whose favorite ester substrate is pNPC8(para-nitrophenyl-caprylate). In order to study thestructure-function relationship of the hyperthermophilic esterases apAPH and AFEST,we exchanged substrate binding domains of the two enzymes, and optimized thenewly formed domain interfaces through site directed mutations. We obtained twofunctional chimeric enzymes PAR and AAM7. Characterization of these two chimericenzymes showed that they inherited the thermophilic properties of the parent proteins.The kinetic parameters indicated that, the esterase substrate specificities of PAR andAAM7were similar to the parent which provided them with the substrate bindingdomain, respectively. Our experimental results showed that: a, the substrate-bindingdomains play a dominant role for the enzyme substrate specificity; b, the optimizationof newly formed domain interface is an important guarantee for obtaining correctlyfolded and stable chimeric enzymes constructed by domain swapping of parents withlow sequence identity.
In one superfamily, proteins can differ greatly in their primary amino acidsequences and in their biological functions. Traces of ancient functional amino acidsequence motifs and structural motifs are, however, often detectable, suggesting thatat least some members of a protein superfamily may be derived from one or morecommon ancestors. By comparing the sequence and structure homologues of theα/β-hydrolase fold superfamily, we discovered the existence of key motifs, which areconserved amino acid sequences within a conserved structural motif. Key motifsrepresent a localized similar structural environment in distant proteins within a proteinsuperfamily. Here, we explored the idea of recombining distant sequence proteins atkey motif regions to engineer novel enzymes. We developed a new domainrecombination strategy-Key Motif Directed Recombination (KMDR). ThroughKMDR, We designed and constructed chimeric proteins from three catalyticallydistinct members of the α/β hydrolase fold superfamily: two hyperthermophilicesterases (AFEST and apAPH) and a mesophilic lipase (Lip1from Candida rugosa)with less than20%sequence identity. The chimeras LAf and LAp retained thesubstrate preference of Lip1for long acyl chain ester substrates and the thermophilic property of the parent AFEST and apAPH.
Recruiting evolved structural domains or subdomains with desired functions frommembers of a superfamily provides a powerful approach for generating largefunctional leaps. As an innovation for domain recombination at inner, rigid areas witha key motif, rather than flexible regions between domains, KMDR thus represents apractical method for the efficient manipulation and recombination of structuraldomains with distantly related sequences. The ability to efficiently design proteinscombining distantly related sequences provides a means to create new biocatalyststhat are useful for fundamental research, and accelerating the commercialization ofvarious products, from pharmaceuticals to fine chemicals. Our work not only shedlight on the efficient recombination of very dissimilar protein sequences within asuperfamily, but also provided the first evidence that key motifs may have playedimportant roles in natural protein evolution.
（一）传统结构域重组结合结构域界面优化获得嵌合酶：来自超嗜热矿泉古菌Aeropyrum pernix K1的嗜热酯酶apAPH和来自嗜热古菌Archaeoglobusfulgidus的嗜热酯酶AFEST具有相似的催化结构域，而它们的底物结合结构域差异较大。apAPH偏好水解中等长度脂肪酸链的酯底物，如pNPC8；AFEST偏好水解短脂肪酸链的酯底物，如pNPC4。为了研究apAPH和AFEST的结构功能关系，我们交换了这两个酶的底物结合结构域，并对新形成的结构域界面进行优化获得了两个有活力的嵌合酶PAR和AAM7。对这两个嵌合酶的性质表征结果表明它们都继承了亲本蛋白质的嗜热性和热稳定性。酶催化动力学结果表明，PAR和AAM7对酯酶底物的链长特异性分别与向它们提供底物结合结构域的亲本一致。我们的实验结果表明：一，底物结合结构域对于酶的底物特异性具有决定性作用；二，低同源性亲本蛋白质进行结构域重组时，对新形成的结构域界面进行优化是获得正确折叠的嵌合酶以及提高嵌合酶稳定性的重要保证。
（二）关键基序指导的结构域重组（KMDR）：同一超家族中的蛋白质成员之间具有相似的折叠结构，但在一级序列和生物功能上可能有很大的差异。在某些成员中可以检测到的蛋白质序列基序和结构基序的存在表明这些成员可能是从一个或多个共同的祖先进化来的。通过对α/β水解酶超家族成员的序列和结构的比对，我们发现了关键基序的存在。关键基序是指存在于保守的结构基序中的保守的序列基序。关键基序代表了蛋白质超家族中成员的相似的局部结构环境。我们设想可以在关键基序区域选择重组位点，利用关键基序的保守性和它与邻近结构的相互作用，重组不同亲本的结构域构建新的嵌合酶。我们提出了一个新的结构域重组的方法—关键基序指导的结构域重组（KMDR）。利用KMDR，以来自于α/β水解酶超家族的三个成员—嗜热酯酶AFEST，嗜热酯酶apAPH和中温脂肪酶Lip1（来源于褶皱假丝酵母Candida rugosa）—为亲本，设计并构建了嗜热的嵌合酶LAf和LAp。对嵌合酶的性质测定结果表明，与我们的预期一致，嵌合酶获得了嗜热亲本AFEST和apAPH的嗜热性和热稳定性，同时也保持了中温脂肪酶亲本Lip1对长链底物的偏好性。我们通过定点突变对嵌合酶LAp进行优化，获得了活力提高4.6倍的突变体。
蛋白质超家族成员中对所需功能的相关结构域进行重组是产生功能跃迁以及扩大合成蛋白质种类的有效途径。作为结构域重组的新方法，KMDR在处于刚性的结构域内部的关键基序区域进行结构域重组，而不是传统重组方法选择的结构域间的柔性连接区域。它有效地重组低同源亲本蛋白质，构建新功能嵌合蛋白质，这对于基础酶学研究以及构建在医药、化工等产业用酶都具有重要意义。我们的研究工作不仅发展了蛋白质超家族中低同源亲本结构域重组的方法，而且首次提出了关键基序在自然界蛋白质进化过程中可能具有重要作用。
Constructing enzymes with desired properties is a major goal for proteinengineers. Recombination is an important mechanism for natural evolution of proteins,and presents an effective strategy for exploring protein sequence space. Diversefunctional domains in proteins provide a resource for designing novel biocatalysts.The recombination of functional protein modules, such as domains or subdomains,from diverse homologous enzymes by conventional domain recombination has led tochimeric enzymes with novel activities, altered substrate specificities, and improvedstability. Recombining proteins with less sequence similarity would offer betteropportunities for creating novel proteins. The challenge has been that the domainsfrom parental proteins with less sequence identity often have structurallynon-compatible interfaces when they are recombined, resulting in inactive chimeras.Thus, further advances in rational protein engineering require a profoundunderstanding of how proteins evolve and new strategies for the efficient generationof chimeras. In this work, we constructed chimeras through two different strategies:traditional domain recombination combining with domain interface refinement; andKey Motif Directed Recombination.
AFEST is a carboxylesterase from Archaeoglobus fulgidus, hydrolyzesp-nitrophenyl esters (pNP) with short acyl chain lengths, and shows the highestactivity towards pNPC4(para-nitrophenyl-butyrate). apAPH is an acylpeptidehydrolase from Aeropyrum pernix K1(apAPH) and shows a promiscuous esteraseactivity with a preference for middle chain length substrates, whose favorite ester substrate is pNPC8(para-nitrophenyl-caprylate). In order to study thestructure-function relationship of the hyperthermophilic esterases apAPH and AFEST,we exchanged substrate binding domains of the two enzymes, and optimized thenewly formed domain interfaces through site directed mutations. We obtained twofunctional chimeric enzymes PAR and AAM7. Characterization of these two chimericenzymes showed that they inherited the thermophilic properties of the parent proteins.The kinetic parameters indicated that, the esterase substrate specificities of PAR andAAM7were similar to the parent which provided them with the substrate bindingdomain, respectively. Our experimental results showed that: a, the substrate-bindingdomains play a dominant role for the enzyme substrate specificity; b, the optimizationof newly formed domain interface is an important guarantee for obtaining correctlyfolded and stable chimeric enzymes constructed by domain swapping of parents withlow sequence identity.
In one superfamily, proteins can differ greatly in their primary amino acidsequences and in their biological functions. Traces of ancient functional amino acidsequence motifs and structural motifs are, however, often detectable, suggesting thatat least some members of a protein superfamily may be derived from one or morecommon ancestors. By comparing the sequence and structure homologues of theα/β-hydrolase fold superfamily, we discovered the existence of key motifs, which areconserved amino acid sequences within a conserved structural motif. Key motifsrepresent a localized similar structural environment in distant proteins within a proteinsuperfamily. Here, we explored the idea of recombining distant sequence proteins atkey motif regions to engineer novel enzymes. We developed a new domainrecombination strategy-Key Motif Directed Recombination (KMDR). ThroughKMDR, We designed and constructed chimeric proteins from three catalyticallydistinct members of the α/β hydrolase fold superfamily: two hyperthermophilicesterases (AFEST and apAPH) and a mesophilic lipase (Lip1from Candida rugosa)with less than20%sequence identity. The chimeras LAf and LAp retained thesubstrate preference of Lip1for long acyl chain ester substrates and the thermophilic property of the parent AFEST and apAPH.
Recruiting evolved structural domains or subdomains with desired functions frommembers of a superfamily provides a powerful approach for generating largefunctional leaps. As an innovation for domain recombination at inner, rigid areas witha key motif, rather than flexible regions between domains, KMDR thus represents apractical method for the efficient manipulation and recombination of structuraldomains with distantly related sequences. The ability to efficiently design proteinscombining distantly related sequences provides a means to create new biocatalyststhat are useful for fundamental research, and accelerating the commercialization ofvarious products, from pharmaceuticals to fine chemicals. Our work not only shedlight on the efficient recombination of very dissimilar protein sequences within asuperfamily, but also provided the first evidence that key motifs may have playedimportant roles in natural protein evolution.
引文
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[1] Pleiss J. Protein design in metabolic engineering and synthetic biology. Curr OpinBiotechnol.2011,22:611-617.
[2] Cheriyan M, Toone EJ, Fierke CA. Improving upon nature: active site remodelingproduces highly efficient aldolase activity toward hydrophobic electrophilic substrates.Biochemistry.2012,51:1658-1668.
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[4] Huang J, Koide A, Makabe K, Koide S. Design of protein function leaps bydirected domain interface evolution. Proc Natl Acad Sci USA.2008,105:6578-6583.
[5] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity ofprogressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice. Nucleic Acids Res.1994,22:4673-4680.
[6] Eswar N, Marti-Renom MA, Webb B, Madhusudhan MS, Eramian D, Shen M,Pieper U, Sali A. Comparative protein structure modeling with MODELLER. CurrProtoc Bioinformatics, John Wiley&Sons, Inc., Supplement15,5.6.1-5.6.30,2006.
[7] Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: aprogram to check the stereochemical quality of protein structures. J App Cryst.1993,26:283.
[8] Luthy R, Bowie JU, Eisenberg D. Assessment of protein models withthree-dimensional profiles. Nature.1992,356:83-85.
[9] D'Amico S, Marx JC, Gerday C, Feller G. Activity-stability relationships inextremophilic enzymes. J Biol Chem.2003,278:7891-7896.
[10] Sreerama N, Woody RW. Computation and analysis of protein circular dichroismspectra. Methods Enzymol.2004,383:318-351.
[11] Sobolev V, Sorokine A, Prilusky J, Abola EE, Edelman M. Automated analysis ofinteratomic contacts in proteins. Bioinformatics.1999,15:327-332.
[12] Wallace AC, Laskowski RA, and Thornton JM. LIGPLOT: a program to generateschematic diagrams of protein-ligand interactions. Protein Eng.1995,8:127-134.
[13] Dundas J, Ouyang Z, Tseng J, Binkowski A, Turpaz Y, and Liang J. CASTp:computed atlas of surface topography of proteins with structural and topographicalmapping of functionally annotated residues. Nucleic Acids Res.2006,34:W116-W118.
[1] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity ofprogressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice. Nucleic Acids Res.1994,22:4673-4680.
[2] Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW,Noble WS. MEME suite: tools for motif discovery and searching. Nucleic Acids Res.2009,37: W202-208.
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