干旱区生态环境知识资源中心(Arid): Directed Evolution of Canonical Loops and Their Swapping between Unrelated Serine Proteinase Inhibitors Disprove the Interscaffolding Additivity Model

Arid

DOI	10.1016/j.jmb.2018.12.003
	Directed Evolution of Canonical Loops and Their Swapping between Unrelated Serine Proteinase Inhibitors Disprove the Interscaffolding Additivity Model
	Boros, Eszter 1; Sebak, Fanni 2,3; Heja, David 1,5; Szakacs, David 1; Zboray, Katalin 1,6; Schlosser, Gitta 4; Micsonai, Andras 1; Kardos, Jozsef 1; Bodor, Andrea 2; Pal, Gabor 1
通讯作者	Pal, Gabor
来源期刊	JOURNAL OF MOLECULAR BIOLOGY
ISSN	0022-2836
EISSN	1089-8638
出版年	2019
卷号	431 期号:3 页码:557-575
英文摘要	Reversible serine proteinase inhibitors comprise 18 unrelated families. Each family has a distinct representative structure but contains a surface loop that adopts the same, canonical conformation in the enzyme inhibitor complex. The Laskowski mechanism universally applies for the action of all canonical inhibitors independent of their scaffold, but it has two nontrivial extrapolations. Intrascaffolding additivity states that all enzyme-contacting loop residues act independently of each other, while interscaffolding additivity claims that these residues act independently of the scaffold. These theories have great importance for engineering proteinase inhibitors but have not been comprehensively challenged. Therefore, we tested the interscaffolding additivity theory by hard-randomizing all enzyme-contacting canonical loop positions of a Kazal- and a Pacifastin-scaffold inhibitor, displaying the variants on M13 phage, and selecting the libraries on trypsin and chymotrypsin. Directed evolution delivered different patterns on both scaffolds against both enzymes, which contradicts interscaffolding additivity. To quantitatively assess the extent of non-additivity, we measured the affinities of the optimal binding loop variants and their binding loop-swapped versions. While optimal variants have picomolar affinities, swapping the evolved loops results in up to 200,000-fold affinity loss. To decipher the underlying causes, we characterized the stability, overall structure and dynamics of the inhibitors with differential scanning calorimetry, circular dichroism and NMR spectroscopy and molecular dynamic simulations. These studies revealed that the foreign loop destabilizes the lower-stability Pacifastin scaffold, while the higher-stability Kazal scaffold distorts the foreign loop. Our findings disprove interscaffolding additivity and show that loop and scaffold form one integrated unit that needs to be coevolved to provide high-affinity inhibition. (C) 2018 Elsevier Ltd. All rights reserved.
英文关键词	directed evolution phage display molecular recognition protein protein interaction SPINK1
类型	Article
语种	英语
国家	Hungary ; USA
收录类别	SCI-E
WOS记录号	WOS:000458222700008
WOS关键词	SECRETORY TRYPSIN-INHIBITOR ; OVOMUCOID 3RD DOMAINS ; MAGNETIC-RESONANCE RELAXATION ; BOVINE ALPHA-CHYMOTRYPSIN ; AMINO-ACID-SEQUENCES ; PHAGE-DISPLAY ; PROTEASE INHIBITORS ; ACTIVE-SITE ; SECONDARY STRUCTURE ; DESERT LOCUST
WOS类目	Biochemistry & Molecular Biology
WOS研究方向	Biochemistry & Molecular Biology
资源类型	期刊论文
条目标识符	http://119.78.100.177/qdio/handle/2XILL650/217238
作者单位	1.Eotvos Lorand Univ, Dept Biochem, Pazmany Peter Setany 1-C, H-1117 Budapest, Hungary; 2.Eotvos Lorand Univ, Inst Chem, Lab Struct Chem & Biol, Pazmany Peter Setany 1-A, H-1117 Budapest, Hungary; 3.Semmelweis Univ, Doctoral Sch Pharmaceut Sci, Ulloi Ut 26, H-1085 Budapest, Hungary; 4.Eotvos Lorand Univ, Inst Chem, Dept Analyt Chem, Pazmany Peter Setany 1-A, H-1117 Budapest, Hungary; 5.Icahn Sch Med Mt Sinai, Dept Med, Translat Transplant Res Ctr, New York, NY 10029 USA; 6.Hungarian Acad Sci, Plant Protect Inst, Ctr Agr Res, Martonvasar, Hungary
推荐引用方式 GB/T 7714	Boros, Eszter,Sebak, Fanni,Heja, David,et al. Directed Evolution of Canonical Loops and Their Swapping between Unrelated Serine Proteinase Inhibitors Disprove the Interscaffolding Additivity Model[J],2019,431(3):557-575.
APA	Boros, Eszter.,Sebak, Fanni.,Heja, David.,Szakacs, David.,Zboray, Katalin.,...&Pal, Gabor.(2019).Directed Evolution of Canonical Loops and Their Swapping between Unrelated Serine Proteinase Inhibitors Disprove the Interscaffolding Additivity Model.JOURNAL OF MOLECULAR BIOLOGY,431(3),557-575.
MLA	Boros, Eszter,et al."Directed Evolution of Canonical Loops and Their Swapping between Unrelated Serine Proteinase Inhibitors Disprove the Interscaffolding Additivity Model".JOURNAL OF MOLECULAR BIOLOGY 431.3(2019):557-575.