Conformation Analysis of Peptides Derived from

Laminin Alpha 1-2 Chain Using Molecular

Dynamics Simulation

Hironao YAMADA, Masaki FUKUDA, Takeshi MIYAKAWA, Ryota MORIKAWA, and

Masako TAKASU

School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0355,

Japan

E-mail: s077112@toyaku.ac.jp

(Received July 15, 2013)

Laminin is one of the components of the basement membrane and has diverse biological

activities. Several functional peptides (EF1-EF5) are identified from LG4 modules of

laminin alpha 1-5 chains. Thus, we perform conformation analysis of EF1 and EF2 using

molecular dynamics simulations. In this study, we perform structure sampling with NPT

ensemble (300 K, 1 bar). Our results show that EF1 peptide has β-sheet structure in water,

and EF2 peptide does not have. Likewise, the EF2 peptide has unstable structure compared

with the EF1 peptide in water.

KEYWORDS: Molecular dynamics simulation, Laminin, Peptide, Conformation

and activity

1. Introduction

The laminins are contained in the basement membrane which exists in various tissues

throughout the body, between epidermis and dermis, muscle, around peripheral nerve and adipocyte. The basement membrane is extracellular matrix which is made of laminin, type IV collagen, perlecan and nidogen. Also, the basement membrane gives the stability of an organization, and has diverse biological activities such as cell adhesion, invasion, proliferation, and differentiation [1-2]. The basement membrane is vital biological material. The laminins play mainly a role of biological activities for functions of the basement membrane. Laminins are giant glycoproteins consisting of three subunits (α, β and γ chains), and five kinds of α chains, three kinds of β chains and three kinds of γ chains have been identified [1-2]. Presently, at least 19 isoforms (laminin 1-19) have been identified by the combination of three subunits [3]. Laminin is functional protein that has diverse biological activities, such as promotion of cell attachment, cell migration, tumor metastasis, neurite outgrowth and angiogenesis [2]. Five laminin-like globular module (LG1-5), where C-terminal region of laminin α chains exists, plays a critical role for the biological activities.

Previously, Suzuki et al. identified several peptides, which have biological functions, in the loop region of the laminin α1-5 chains LG4 module [4]. The EF1 peptide

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JPS Conf. Proc. , 016016 (2014)©2014 The Physical Society of Japan

1Proceedings of the 12th Asia Pacific Physics Conference

016016-1

(DYATLQLQEGRLHFMFDLG, human α1 chain residues 2762-2780) promotes α2β1 integrin mediated cell attachment. The sEF1 peptide (LQLQEGRLHFMFD), which is a truncated peptide of EF1, has lower activities than EF1. Previous research also showed that biological activities of sEF1 are enhanced by disulfide bond. Accordingly, the importance of looped structure was suggested [4]. On the other hand, EF2 peptide (DFATVQLRNGFPYFSYDLG, mouse α2 chain residues 2808-2826), which is homological sequences of EF1 peptide, does not have biological activities [5]. Formerly, we performed conformation analysis of peptides (EF1, sEF1 and EF2) in LG4 modules using molecular dynamics simulations. Our previous results showed that EF1 has hairpin-like structure (EF2 does not have), and we indicated relation between activities and the structure [6-7]. In this paper, we investigate dynamics behavior of EF1 and EF2 peptides at 300K and 1bar (NPT ensemble).

2. Methods and Results

2.1 Simulation methods and analysis

We prepared the EF2 peptide from the structure of mouse laminin α2 chain LG4 module determined by an X-ray diffraction (PDB ID: 1DYK) [8]. The EF1 peptide is obtained by amino acid substitution for EF2. Molecular dynamics simulation with NPT ensemble is performed for 500 ns. We calculate RMS-deviation (RMSD) and RMS-fluctuation (RMSF) as a measure. Conformation of EF2 peptide in protein structure has β-hairpin (Fig. 1). Then, we measure the length of hydrogen bond (H-bond) for EF1 and EF2 peptides obtained by NPT ensemble (Fig. 2)

2.2 Results

We show RMSD (Fig. 3) and RMSF (Fig. 4) of EF1 and EF2 peptides obtained under NPT ensemble. These values of RMSD show similar tendency. However, the maximum peak value of RMSD of EF2 was larger compared with the value of EF1. These results indicate that EF1 has stable conformation relative to EF2.

Fig. 1. Conformation of α2LG4 module. Black

color strand indicates the EF2 peptide sequences,

showing β-hairpin structure.

Fig. 3. RMSD (Backbone) with respect

to EF1 and EF2 peptides obtained by

NPT ensemble. Black and grey colors

indicate RMSD of the EF1 and EF2.

α β γ Ζ* Ζ γ β α EF1 DYATLQLQEGRLHFMFDLG

EF2 DFATVQLRNGFPYFSYDLG Fig. 2. Amino acid sequences of EF1 and EF2

peptides. α, β, γ and Ζ are points for

measuring the length of H-bond.

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016016-2JPS Conf. Proc. , 016016 (2014)1

In Fig. 4, we present our results of RMSF (backbone) for EF1 and EF2 peptides obtained by simulation. The RMSF of EF2 peptide is large compared with fluctuation of EF1. This result indicates that the EF1 peptide has stable structure. In contrast, the EF2 peptide has unstable structure compared with the EF1. Fluctuations in the neighborhoods of C-terminal and N-terminal are also large for EF1 and EF2 peptide. In other words, it indicates that, in the neighborhoods of C-terminal and N-terminal of EF1 and EF2 peptides, β-sheet structure is not found.

We obtained conformations of EF1 and EF2 peptides after 500 ns, and we also calculated the averaged conformations of EF1 and EF2 peptides. In Fig. 5, we show these conformations. Conformation of EF1 after 500 ns has hairpin–like structure, and EF2 does not have. Although EF1 peptide after simulation has β-sheet structure, EF2 peptide does not have. Thus, this result indicates that the EF2 peptide has unstable structure compared with the EF1 peptide.

In Fig. 6, we show the length of H-bond needed to maintain β-sheet structure. Among eight H-bonds, five H-bonds have larger value for EF2 than for EF1. This result suggests that, in the neighborhoods of C-terminal and N-terminal, H-bond was not found, and we verify that EF1 has five H-bonds. Also, we observed that H-bond of EF2 peptide forΖ was not formed for a

(a) (b) (c) (d)

Fig. 5. Conformations of EF1 and EF2 peptides. (a) and (b)

are after 500 ns. (c) and (d) are averaged conformations. (a)

and (c) are EF1 peptide and (b) and (d) are EF2 peptide.

α-Ⅰ α-Ⅱ

β-Ⅰ β-Ⅱ

γ-Ⅰ γ-Ⅱ

Ζ-Ⅰ Ζ-Ⅱ

Fig. 6. Length of H-bonds needed for

folding of β-sheet for the EF1 and EF2

peptides. We account for α, β, γ and Ζ on

legends in Fig. 2. Black and grey colors

indicate the EF1 and EF2.

Time (ps)

Dis

tance

(nm

)

Time (ps)

Dis

tance

(nm

)

Time (ps)

Dis

tance

(nm

)

Time (ps)

Dis

tance

(nm

)

Time (ps)

Dis

tance

(nm

)

Time (ps)

Dis

tance

(nm

)

Time (ps)

Dis

tance

(nm

)

Time (ps)

Dis

tance

(nm

)

Fig. 4. RMSF (backbone) with respect

to EF1 and EF2 peptides obtained by

NPT ensemble. Black and grey colors

indicate RMSD of the EF1 and EF2.

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016016-3JPS Conf. Proc. , 016016 (2014)1

brief moment at around 150 ns. These results show that the EF1 peptide has β-sheet structure and the EF2 peptide does not have.

3. Conclusion

We performed molecular dynamics simulations of the EF1 and EF2 peptides with

NPT ensemble for 500 ns. We calculated RMSD, RMSF and length of H-bonds needed for the folding of β-sheet in regard to the EF1 and EF2 peptides. In the results of RMSD, we found that the EF1 peptide has stable structure compared with the EF2 peptide in water. The result of RMSF shows that fluctuation of the EF1 peptide has smaller value compared with value of the EF2. Also, the EF1 peptide obtained by simulation has β-sheet structure, and the EF2 peptide has no β-sheet structure. This result can be confirmed from the calculation of H-bond length. In conclusion, our results indicate that the difference of conformation between the EF1 and EF2 peptides is related to the biological activity.

Acknowledgment

This work was supported by JSPS KAKENHI Grant Number 24540442.

References

[1] T. Sasaki, R. Fassler and E. Hohenester: J. Cell. Biol. 164 (2004) 959-963.

[2] H. Colognato and P. D. Yurchenco: Dev. Dyn. 218 (2000) 213-234.

[3] D. Ramadhani, T. Tsukada, K. Fujiwara, K. Horiguchi, M. Kikuchi and T. Tashiro: Acta Histochem.

Cytochem. 45(5) (2012) 309–315.

[4] N. Suzuki, H. Nakatsuka, M. Mochizuki, N. Nishi, Y. Kadoya, A. Utani, S. Oishi, N. Nobutaka, H.

K. Kleinman, and M. Nomizu: J. Biol Chem. 278 (2003) 45697-45705.

[5] F. Katagiri, T. Hara, Y. Yamada, S. Urushibata, K. Hozumi, Y. Kikkawa, and M. Nomizu: Peptide

Science 2008. (2009) 243-246.

[6] H. Yamada, Y. Komatsu, T. Miyakawa, R. Morikawa, F. Katagiri, K. Hozumi, Y. Kikkawa, M.

Nomizu, and M. Takasu: Peptide Science 2011. (2012) 201-204.

[7] H. Yamada, T. Miyakawa, R. Morikawa, F. Katagiri, K. Hozumi, Y. Kikkawa, M. Nomizu, and

M. Takasu: Peptide Science 2012. (2013) 339-342.

[8] D. Tisi, J. Talts, R. Timple, and E. Hohenester: EMBO. J. 19 (2000) 14732-1440.

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