基于胶原异三聚体的成骨不全症机理研究

doi:10.3969/j.issn.1000-5641.2023.06.010

摘要/Abstract

摘要：

胶原蛋白是细胞外基质的主要成分, 由3条链缠绕构成三螺旋. 在28种天然胶原中占比最大的是Ⅰ型胶原蛋白, 它是由两条α1链与一条α2链构成的异三聚体, α1或α2链中甘氨酸单点突变会导致成骨不全症. 基于更接近天然胶原的异三聚体模型(abc), 3条链中分别引入Gly→Ala, 构建7种突变体. 差示扫描量热结果表明, 单点突变体的熔融温度(T_m)值降低15℃, 双点及三点突变体未形成三螺旋结构. 利用梯阶模型分析分子动力学模拟轨迹, 突变点附近梯阶参数值发生变化, 表明三螺旋结构局部解折叠. 引入弹性函数量化胶原结构变化程度, 发现氢键能量与结构形变分数具有高关联性(R²= 0.76), 表明突变不仅破坏了氢键作用力, 也导致了分子的弯曲与运动状态的变化. 结合计算与实验, 解析了甘氨酸突变对胶原整体结构与运动模式的影响, 为进一步揭示甘氨酸突变的致病机理提供了理论基础.

关键词: 胶原蛋白, 异三聚体, 梯阶模型, 结构形变, 成骨不全症

Abstract:

In this study, Gly→Ala was introduced into three chains of the heterotrimeric model (abc); seven mutants were subsequently constructed, and the local structure and global motion changes were analyzed. DSC results showed that the T_m value of the single point mutation was reduced by about 15°C, while the double and triple point mutations did not form triple helical structures. MD simulation trajectories were analyzed by ladder models; the results showed that the value of the step parameter changes near the mutation point, indicating an unfolding of the triple helix structure. An elastic function was introduced to quantify the degree of collagen structure change. It was found that the hydrogen bond energy was highly correlated with the structural deformation fraction ( $ R^2=0.76 $ ), indicating that the mutation not only destroyed the hydrogen bond force, but also resulted in changes in the bending and motion states of the molecule. This study, combined with calculations and experiments, helped quantify the effects of glycine mutation on the overall structure and movement pattern of collagen. Hence, the study provides a theoretical basis for clarifying the pathogenic mechanism of glycine mutation.

Key words: collagen, heterotrimer, step model, structural deformation, osteogenesis imperfecta

中图分类号:

强书敏, 吕成, 许菲. 基于胶原异三聚体的成骨不全症机理研究[J]. 华东师范大学学报（自然科学版）, 2023, 2023(6): 108-118.

Shumin QIANG, Cheng LYU, Fei XU. Mechanism of osteogenesis imperfecta based on collagen heterotrimer[J]. Journal of East China Normal University(Natural Science), 2023, 2023(6): 108-118.

图/表 7

图1

表1

图2

图3

图4

图5

图6

参考文献 54

1	MYLLYHARJU J, KIVIRIKKO K I.. Collagens, modifying enzymes and their mutations in humans, flies and worms. Trends Genet, 2004, 20 (1): 33- 43.
2	SHOULDERS M D, RAINES R T.. Collagen structure and stability. Annual Review of Biochemistry, 2009, 78, 929- 958.
3	HEINO J.. The collagen family members as cell adhesion proteins. Bioessays, 2007, 29 (10): 1001- 1010.
4	BRINCKMANN J. Collagens at a Glance [M]// BRINCKMANN J, NOTBOHM H, MÜLLER P K. Collagen: Primer in Structure, Processing and Assembly. Berlin: Springer, 2005: 1-6.
5	RAMACHANDRAN G N, SASISEKHARAN V.. Structure of Collagen. Nature, 1961, 190 (4780): 1004- 1005.
6	RICH A, CRICK F H C.. The molecular structure of collagen. Journal of Molecular Biology, 1961, 3 (5): 483- 506.
7	MYLLYHARJU J, KIVIRIKKO K I.. Collagens and collagen-related diseases. Annals of Medicine, 2001, 33 (1): 7- 21.
8	KUIVANIEMI H, TROMP G, PROCKOP D J.. Mutations in fibrillar collagens (types Ⅰ, Ⅱ, Ⅲ, and Ⅹ Ⅰ), fibril-associated collagen (type Ⅰ Ⅹ), and network-forming collagen (type Ⅹ) cause a spectrum of diseases of bone, cartilage, and blood vessels. Human Mutation, 1997, 9 (4): 300- 315.
9	MARINI J C, FORLINO A, CABRAL W A, et al.. Consortium for osteogenesis imperfecta mutations in the helical domain of type Ⅰ collagen: Regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans. Human Mutation, 2007, 28 (3): 209- 221.
10	BECK K, CHAN V C, SHENOY N, et al.. Destabilization of osteogenesis imperfecta collagen-like model peptides correlates with the identity of the residue replacing glycine. Proceedings of the National Academy of Sciences, 2000, 97 (8): 4273- 4278.
11	BAUM J, BRODSKY B.. Structural biology: Modelling collagen diseases. Nature, 2008, 453 (7198): 998.
12	BRODSKY B, PERSIKOV A V.. Molecular structure of the collagen triple helix. Advances in Protein Chemistry and Structural Biology, 2005, 70, 301- 339.
13	FIELDS G B.. A model for interstitial collagen catabolism by mammalian collagenases. Journal of Theoretical Biology, 1991, 153 (4): 585- 602.
14	OKUYAMA K, HONGO C, FUKUSHIMA R, et al.. Crystal structures of collagen model peptides with Pro-Hyp-Gly repeating sequence at 1.26 A resolution: implications for proline ring puckering. Biopolymers, 2004, 76 (5): 367- 377.
15	GOODMAN M, BHUMRALKAR, JEFFERSON E A, et al.. Collagen mimetics. Biopolymers, 1998, 47 (2): 127- 142.
16	BAUM J, BRODSKY B. Folding of peptide models of collagen and misfolding in disease [J]. Current Opinion in Structural Biology, 1999, 9(1): 122-128.
17	BELLA J.. A new method for describing the helical conformation of collagen: Dependence of the triple helical twist on amino acid sequence. Journal of Structural Biology, 2010, 170 (2): 377- 391.
18	OKUYAMA K.. Revisiting the molecular structure of collagen. Connective Tissue Research, 2008, 49 (5): 299- 310.
19	PERSIKOV A V, RAMSHAW J A, KIRKPATRICK A, et al.. Amino acid propensities for the collagen triple-helix. Biochemistry, 2000, 39 (48): 14960- 14967.
20	PERSIKOV A V, RAMSHAW J A M, BRODSKY B.. Prediction of collagen stability from amino acid sequence. Journal of Biological Chemistry, 2005, 280 (19): 19343- 19349.
21	RAMSHAW J A M, SHAH N K, BRODSKY B.. Gly-X-Y tripeptide frequencies in collagen: A context for host–guest triple-helical peptides. Journal of Structural Biology, 1998, 122 (1): 86- 91.
22	MANJIRI, BHATE, XIN, et al.. Folding and conformational consequences of glycine to alanine replacements at different positions in a collagen model peptide. Biochemistry, 2002, 41 (20): 6539- 6547.
23	BODIAN D L, MADHAN B, BRODSKY B, et al.. Predicting the clinical lethality of osteogenesis imperfecta from collagen glycine mutations. Biochemistry, 2008, 47 (19): 5424- 5432.
24	BERG R A, PROCKOP D J.. The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen. Biochemical and Biophysical Research Communications, 1973, 52 (1): 115- 120.
25	BELLA J, EATON M, BRODSKY B, et al.. Crystal and molecular structure of a collagen-like peptide at 1.9 Å resolution. Science, 1994, 266 (5182): 75- 81.
26	LI Y, BRODSKY B, BAUM J.. NMR conformational and dynamic consequences of a gly to ser substitution in an osteogenesis imperfecta collagen model peptide. Journal of Biological Chemistry, 2009, 284 (31): 20660- 20667.
27	O'LEARY L E R, FALLAS J A, HARTGERINK J D.. Positive and negative design leads to compositional control in AAB collagen heterotrimers. Journal of The American Chemical Society, 2011, 133 (14): 5432- 5443.
28	XIAO J, SUN X, MADHAN B, et al.. NMR studies demonstrate a unique AAB composition and chain register for a heterotrimeric type Ⅳ collagen model peptide containing a natural interruption site*. Journal of Biological Chemistry, 2015, 290 (40): 24201- 24209.
29	JALAN A A, HARTGERINK J D.. Simultaneous control of composition and register of an AAB-type collagen heterotrimer. Biomacromolecules, 2013, 14 (1): 179- 185.
30	XU F, ZAHID S, SILVA T, et al.. Computational design of a collagen A: B: C-type heterotrimer. Journal of the American Chemical Society, 2011, 133 (39): 15260- 15263.
31	CLEMENTS K A, ACEVEDO-JAKE A M, WALKER D R, et al.. Glycine substitutions in collagen heterotrimers alter triple helical assembly. Biomacromolecules, 2017, 18 (2): 617- 624.
32	ACEVEDO-JAKE A M, CLEMENTS K A, HARTGERINK J D.. Synthetic, register-specific, AAB heterotrimers to investigate single point Glycine mutations in osteogenesis imperfecta. Biomacromolecules, 2016, 17 (3): 914- 921.
33	PARK S, KLEIN T E, PANDE V S.. Folding and misfolding of the collagen triple helix: Markov analysis of molecular dynamics simulations. Biophysical Journal, 2007, 93 (12): 4108- 4115.
34	MOONEY S D.. Structural models of osteogenesis imperfecta-associated variants in the COL1A1 gene. Molecular & Cellular Proteomics, 2002, 1 (11): 868- 875.
35	BODIAN D L, RADMER R J, HOLBERT S, et al.. Molecular dynamics simulations of the full triple helical region of collagen type I provide an atomic scale view of the protein's regional heterogeneity. Pacific Symposium on Biocomputing, 2011, 193- 204.
36	FEI X, ZHENG H, CLAUVELIN N, et al. Parallels between DNA and collagen - Comparing elastic models of the double and triple helix [J]. Scientific Reports, 2017, 7(1): 12802.
37	ZHENG H, LU C, LAN J, et al.. How electrostatic networks modulate specificity and stability of collagen. Proc Natl Acad Sci USA, 2018, 115 (24): 6207- 6212.
38	LINDORFF-LARSEN K, PIANA S, PALMO K, et al.. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins: Structure, Function, and Bioinformatics, 2010, 78 (8): 1950- 1958.
39	PRONK S, PÁLL S, SCHULZ R, et al.. GROMACS 4.5: A high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics, 2013, 29 (7): 845- 854.
40	ZHENGWEI P, EWIG C S, HWANG M J, et al.. Comparison of simple potential functions for simulating liquid water. The Journal of Physical Chemistry A, 1997, 101, 7243- 7252.
41	DARDEN T A, YORK D M, PEDERSEN L G.. Particle mesh Ewald-An N·log(N) method for Ewald sums in large systems. Journal of Computational Chemistry, 1993, 18 (12): 1463- 1472.
42	BERENDSEN H J C P, POSTMA J P M V, GUNSTEREN W F V, et al.. Molecular-dynamics with coupling to an external bath. The Journal of Chemical Physics, 1984, 81, 3684.
43	OLSON W K, BANSAL M, BURLEY S K, et al.. A standard reference frame for the description of nucleic acid base-pair geometry. Journal of Molecular Biology, 2001, 313 (1): 229- 237.
44	OLSON W K.. DNA sequence-dependent deformability deduced from protein-DNA crystal complexes. Proceedings of the National Academy of Sciences, 1998, 95 (19): 11163- 11168.
45	LUZAR A, CHANDLER D.. Effect of environment on hydrogen bond dynamics in liquid water. Physrevlett, 1996, 76 (6): 928.
46	GORDON D B, MARSHALL S A, MAYO S L.. Energy functions for protein design. Current Opinion in Structural Biology, 1999, 9 (4): 509- 513.
47	FU I, CASE D A, BAUM J.. Dynamic water-mediated hydrogen bonding in a collagen model peptide. Biochemistry, 2015, 54 (39): 6029- 6037.
48	BABCOCK M S, OLSON W K.. The effect of mathematics and coordinate system on comparability and "dependencies" of nucleic acid structure parameters . Journal of Molecular Biology, 1994, 237 (1): 98- 124.
49	GAJKO-GALICKA A.. Mutations in type Ⅰ collagen genes resulting in osteogenesis imperfecta in humans. Acta Biochimica Polonica, 2002, 49 (2): 433- 441.
50	KUIVANIEMI H, TROMP G, PROCKOP D J.. Mutations in collagen genes: Causes of rare and some common diseases in humans. Faseb Journal, 1991, 5 (7): 2052- 2060.
51	WALLACE J M, ERICKSON B, LES C M, et al.. Distribution of type Ⅰ collagen morphologies in bone: Relation to estrogen depletion. Bone, 2010, 46 (5): 1349- 1354.
52	LI T, CHANG S W, RODRIGUEZ-FLOREZ N, et al.. Studies of chain substitution caused sub-fibril level differences in stiffness and ultrastructure of wildtype and oim/oim collagen fibers using multifrequency-AFM and molecular modeling. Biomaterials, 2016, 107, 15- 22.
53	XIAO J, MADHAN B, LI Y, et al.. Osteogenesis imperfecta model peptides: Incorporation of residues replacing Gly within a triple helix achieved by renucleation and local flexibility. Biophysical Journal, 2011, 101 (2): 449- 458.
54	XIAO J, CHENG H, SILVA T, et al.. Osteogenesis imperfecta missense mutations in collagen: Structural consequences of a Glycine to alanine replacement at a highly charged site. Biochemistry, 2011, 50 (50): 10771- 10780.

Type	ΔT_m/ ℃	Δ摆动角/ (°)	Δ面积^a/ ?²	Δrise^a/ ?	Δslide^a/ ?	Δshift^a/ ?	Δroll^a/ (o)	Δtilt^a/ (o)	Δtwist^a/ (o)	ΔE_HB^b	ΔE_DS^b	Tilt-Twist^c
Type	ΔT_m/ ℃	Δ摆动角/ (°)	Δ面积^a/ ?²	Δrise^a/ ?	Δslide^a/ ?	Δshift^a/ ?	Δroll^a/ (o)	Δtilt^a/ (o)	Δtwist^a/ (o)	ΔE_HB^b	ΔE_DS^b	倾斜角/(°)	长短轴比
a'bc	15.65	40	0.12	–0.4±0.6	0.4±0.4	–0.5±0.7	–1.43±1.03	2.73±2.43	–4.00±2.81	0.14±0.17	17.39±16.70	43	2.29
ab'c	15.63	38	0.17	–0.4±0.4	–0.2±1.1	–0.6±1.1	–1.93±2.64	2.68±2.54	–4.62±4.93	0.16±0.45	30.18±22.86	42	1.65
abc'	14.77	41	0.11	–0.5±0.2	0.4±1.1	–0.9±0.3	–2.81±1.42	4.18±1.47	–7.12±2.83	0.10±0.29	33.84±23.70	29	1.52
a'b'c		53	0.35	–0.5±0.4	–0.3±1.0	–0.6±1.1	–2.23±2.29	3.61±3.40	–5.79±5.01	0.31±0.39	37.25±25.78
a'bc'		47	0.40	–0.7±0.1	–0.0±0.3	–1.1±0.1	–3.36±1.55	4.56±1.23	–7.98±2.01	0.16±0.31	39.93±18.95
ab'c'		49	0.31	–0.6±0.2	–0.5±0.9	–0.7±0.3	–2.68±1.60	5.24±2.21	–8.81±3.57	0.34±0.35	42.03.±32.33
a'b'c'		54	0.63	–0.4±0.3	0.9±1.2	–0.4±0.5	–3.00±2.25	4.81±0.75	–8.02±2.89	0.35±0.64	43.67±29.22