Structural Basis for the Varying Propensities of Different Amino Acids

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Structural Basis for the Varying Propensities of Different Amino Acids To Adopt the Collagen Conformation S. Sundar Raman ,†,‡ R. Gopalakrishnan,† R. C. Wade,‡ and V. Subramanian*,† † ‡

Chemical Laboratory, Central Leather Research Institute, Council of Scientific and Industrial Research, Adyar, Chennai 600 020, India Molecular and Cellular Modeling Group, Heidelberg Institute for Theoretical Studies (HITS) gGmbH, Schloss-Wolfsbrunnenweg 35, 69118 Heidelberg, Germany

bS Supporting Information ABSTRACT: Although previous experimental studies have shown the positional preference of different amino acids (AAs) to form a stable triple helical collagen motif, the structural basis for the variations in the sequence and the positional propensity has not been systematically investigated. Thus, we have here probed the origin of the structural stability offered by the 20 naturally occurring AAs to collagen by means of classical molecular dynamics (MD) simulation. Simulations were carried out on 39 collagen-like peptides employing a host-guest approach. The results show that the propensity of the different AAs to adopt collagen-like conformations depends primarily on their φ and ψ angle preferences. Changes in these angles upon substitution of different AAs in the XAA and YAA positions in the canonical ((Gly-XAA-YAA)7)3 motif dictate the formation of interchain hydrogen bonds, solvent interactions, and puckering of neighboring imino acids and, thus, the structural stability of the collagen. The role of solvent-mediated hydrogen bonds in the stabilization of collagen has also been elucidated from the MD simulations. In addition to the conventional hydrogen bonds known to be present in collagen, a hitherto unidentified direct interchain hydrogen bond, between the XAA N-H group and the Hyp O-H group of the neighboring chain, was observed during the simulations. Its occupancy was ∼36% when Leu was present at the XAA position.

1. INTRODUCTION Collagens form a family of proteins having a characteristic triple helical structure. They are the most abundant proteins in mammals. Since collagens are structural proteins with unique hierarchical structures and some of them form fibers, they are responsible for the integrity of many tissues in vertebrates.1-4 Molecular defects in collagen genes lead to changes in the collagen triple helical structure which cause diseases such as chondrodysplasias, Osteogenesis imperfecta (OI), Alport syndrome, Ehlers-Danlos syndrome and Epidermolysis bullosa.5-12 OI, for example, arises from substitution of Gly by bulkier amino acids (AA) in the collagen triple helix structure.11-15 The triple helical structure of collagen with two hydrogen bonds for every three residues was proposed 55 years ago by Ramachandran and Kartha.16 At the same time, Rich and Crick presented an alternative triple helical model with one hydrogen bond for every three residues.17 Later, Okuyama et al. further refined the structure using X-ray diffraction data.18 Ever since the structure of collagen was proposed, it has received widespread attention from many researchers. Particularly, studies on the number of hydrogen bonds per amino acid triplet and the stability of the collagen were carried out in the formative years of the structural biology of collagen.19-27 In addition, numerous investigations have been carried out on biophysical and biochemical aspects of collagen.28-35 Even today, the origin of the r 2011 American Chemical Society

high stability of the mummified type I collagen from the 5300year-old “Tyrolean Iceman” has been investigated, vividly demonstrating the importance of collagen research in today’s context.36 The hallmark of collagen is that it is composed of three polypeptide chains. Amino acid (AA) sequence analysis of the triple helical domain of collagen reveals that nearly 33% of its residues are glycine (Gly) and around 25% are proline (Pro). Every third residue in the AA sequence of the triple helical domain is occupied by Gly, leading to a Gly-XAA-YAA repeating pattern or motif. Studies on mature collagen are complicated due to variations in sequence, composition, and length. The complexity can be reduced by the design of appropriate model collagen-like peptides (Clps).15 Hence, Clps have been used to investigate the sequence-dependent structure, folding, and stability of collagen. Sequence analysis of fibril-forming collagen revealed that the occurrence of (Gly-Pro-Hyp) is higher than that of other triplets.37 In addition, previous studies showed that (Gly-ProHyp) confers maximum structural stability to triple helical conformation.15 Brodsky’s group has made pioneering contributions to Received: September 24, 2010 Revised: January 19, 2011 Published: March 01, 2011 2593

dx.doi.org/10.1021/jp109133v | J. Phys. Chem. B 2011, 115, 2593–2607

The Journal of Physical Chemistry B

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Table 1. Melting Temperature and Enthalpy of Triplet Repeat Triple Helices and the Occurrence of These Triplets in Natural Collagen Gly-XAA-Hyp Tm (C)

ΔH (kJ/mol)

occurrence (%)

Pro/Hyp

47.3

435

35.6

Arg

40.6

520

3.0

Lys Asp

41.5 40.1

540 520

3.7 4.1

Glu

42.9

590

His

36.5

580

a

charged

ΔH (kJ/mol)

occurrence (%)

47.3

435

45.1

47.2

610

7.7

36.8 34.0

400 776

7.9 0.8

7.0

39.7

630

1.6

1.0

35.7

497

a

Tm (C)

a

18.8

0.5 18.5

Ser

38.0

506

5.0

35.0

435

4.1

Thr

36.2

506

2.7

39.7

647

1.7

Asn

38.3

502

1.4

30.3

640

0.7

Gln Cys

40.4 36.1

565 423

2.2 0.0

41.3 37.7

559 471

8.2 0.1

Gly

33.2

575

1.4

32.7

665

4.4

Ala

41.7

480

6.4

40.9

502

8.6

Val

38.9

518

3.2

40.0

481

3.1

Met

38.6

452

1.7

42.6

436

2.2

Ile

38.4

624

3.7

41.5

559

1.1

Leu nonpolar

39.0

437

12.7 29.1

31.7

514

1.7 21.1

Phe

33.5

514

4.1

28.3

557

0.2

Trp

31.9

593

0.0

26.1

670

0.0

Tyr

34.3

629

1.1

30.2

657

0.3

polar

11.3

aromatic a

Gly-Pro-YAA

AA

a

14.8

5.2

0.5

Experimental data were taken from ref 44.

the understanding of the sequence, structure, folding, and stability of collagen.37-50 They have employed a host-guest approach to design various Clps. The host-guest Clps were built using (Gly-Pro-Hyp)n as the host and Gly-XAA-YAA as the guest. The melting temperature and enthalpy of host-guest Clps from previous study44 are given in Table 1 along with the percentage of occurrence of various AAs at the XAA and YAA positions in the natural collagen. It is clear from Table 1 that the presence of imino acids in the XAA and YAA positions confers maximum stability to collagen, whereas the occurrence of non-imino acids in the XAA and YAA positions results in variations in global thermal stability and modulates local stability. It is evident from the host-guest peptide studies on collagen that the presence of amino acids such as Pro, Glu, Ala, Lys, Arg, Gln, and Asp in the XAA position results in the most stabilizing Gly-XAA-Hyp triplets. On the other hand, the occurrence of Hyp, Arg, Met, Ile, Gln, and Ala at the YAA position results in the most stable composition for Gly-Pro-YAA triplets. The presence of Gly and other aromatic amino acids in the XAA and YAA positions leads to lower stability.51 For example, It can be seen from Table 1 that when Asp is present at the XAA position, the most stabilizing sequence has a melting temperature of 40.1 C, whereas the same sequence with Asp in the YAA position has a melting temperature of 34.0 C. In the case of aromatic residues, the melting temperature of the sequence with the triplet Gly-Phe-Hyp is 33.5 C, whereas the sequence with Gly-Pro-Phe has a melting temperature of 28.3 C. Thus it is necessary to probe the origin of the thermal

and structural stability (destability) provided by each amino acid for the formation of the triple helical motif.52-58 Schematic representations of the host-guest Clps that are used in the present investigation are shown in Table 2 along with their AA compositions. In this investigation, the molecular basis for the origin of the stability of the 20 naturally occurring guest AAs at the XAA and also at the YAA (except Pro) positions has been probed using the host-guest approach and employing classical MD simulations.

2. MATERIALS AND METHODS 2.1. Model Building and MD Simulation Details. Triple helical structures were built for the host-guest Clps using the Gencollagen package.59 The 20 naturally occurring AAs were used as guest residues at the XAA position in the host system of Gly-Pro-Hyp repeats. Similarly, the same AAs except Pro were used as guest residues at the YAA position in the host system of the Gly-Pro-Hyp repeats. The sequences of all three polypeptide chains were identical (see Table 2). In the models with guest at XAA, the 12th residues of the chains A, B, and C are replaced by the different AAs. In the case of guest at YAA, the 13th residues of the chains A, B, and C are substituted by various AAs. The force field developed by Park et al. was used for 4Rhydroxyproline,60 and for the other AAs, AMBER99SB61 was used. All-atom molecular dynamics (MD) simulations were 2594

dx.doi.org/10.1021/jp109133v |J. Phys. Chem. B 2011, 115, 2593–2607


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Table 2. Amino Acid Sequence Composition of the Host-Guest Collagen-like Peptides Studied in This Investigation chain models with guest at XAA position no. of each chain

triad no.

A

B

models with guest at YAA C

A

B

C

1

Ace

2

Gly

Ace

Ace Gly

Ace

3

Pro

Gly

Ace

Pro

Gly

Ace

Pro

Gly

Hyp

Pro

Gly

4

1

Hyp

5

2

Gly

Hyp

Pro

Gly

Hyp

Pro

6

3

Pro

Gly

Hyp

Pro

Gly

Hyp

7

4

Hyp

Pro

Gly

Hyp

Pro

Gly

8 9

5 6

Gly Pro

Hyp Gly

Pro Hyp

Gly Pro

Hyp Gly

Pro Hyp

10

7

Hyp

Pro

Gly

Hyp

Pro

Gly

11

8

Gly

Hyp

Pro

Gly

Hyp

Pro

12

9

XAA

Gly

Hyp

Pro

Gly

Hyp

13

10

Hyp

XAA

Gly

YAA

Pro

Gly

14

11

Gly

Hyp

XAA

Gly

YAA

Pro

15

12

Pro

Gly

Hyp

Pro

Gly

YAA

16 17

13 14

Hyp Gly

Pro Hyp

Gly Pro

Hyp Gly

Pro Hyp

Gly Pro

18

15

Pro

Gly

Hyp

Pro

Gly

Hyp

19

16

Hyp

Pro

Gly

Hyp

Pro

Gly

20

17

Gly

Hyp

Pro

Gly

Hyp

Pro

21

18

Pro

Gly

Hyp

Pro

Gly

Hyp

22

19

Hyp

Pro

Gly

Hyp

Pro

Gly

Nme

Hyp

Pro

Nme

Hyp

Pro

Nme

Hyp Nme

Nme

Hyp Nme

were performed for each system at 300 K in the NPT ensemble. The trajectories were saved every picosecond for further analysis. 2.2. Analysis of the MD Simulations. 2.2.1. Radius of Gyration (Rg). Rg is a parameter that describes the mean extent of a system64 and is computed in two steps. First, the coordinates of the center of mass Rc are determined. Then Rg at time t is determined from the following equation: P mi ðri ðtÞ - Rc ðtÞÞ P ð1Þ Rg ðtÞ ¼ mi where mi is the mass of the ith atom and ri its coordinates. Rg per residue for each polypeptide chain was calculated in this investigation. 2.2.2. Root Mean Square Deviation. The backbone root mean square deviation (rmsd) was calculated with respect to initial conformation as a function of time using eq 2, where ri is the vector of atom i at time t; ri(0) is the initial vector of the same atom; and N is the total number of backbone atoms (N, CR, C, and O).

rmsdðtÞ ¼ 2595

N 1 X ½ ri ðtÞ - ri ð0Þ 2 1=2 N i¼1

)

carried out using the AMBER10 package (http://ambermd. org/). The molecular dynamics simulations were performed employing periodic boundary conditions and an explicit solvent environment (TIP3P62 water model) with a minimum distance of 12.0 Å from the solute to the edge of the solvent box. The boxes had dimensions of approximately 5 5 10 nm3 and contained 4470-7797 water molecules. Naþ and Cl- counterions were used to neutralize the systems. The particle mesh Ewald method was used for the calculation of the electrostatic interactions, and the cutoff for the nonbonded interactions was set as 12 Å. The entire system was minimized with 1000 cycles of steepest decent followed by 10 000 cycles of conjugate gradient minimization. The system was annealed from 0 to 300 K over 70 ps using a time step of 1 fs with the peptide atoms restrained by a harmonic potential of 10 (kcal/mol)/Å2. The SHAKE algorithm was used to constrain bonds involving hydrogen atoms.63 Water molecules were then equilibrated for 50 ps in the NPT ensemble while retaining the harmonic restraints on the peptide atoms. The pressure of the system was kept at 1 atm using isotropic position scaling (pressure regulation procedure) with a time relaxation of 1 ps. The temperature of the system was regulated using the Langevin dynamics algorithm with a collision frequency γ = 2 ps-1, which is recommended for the Amber package. The systems were then equilibrated without restraints on any atoms in the NPT ensemble at 300 K for 1.5 ns. Finally, production runs of 3 ns

)

23

ð2Þ



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2.2.3. Diameter of the Triple Helix. During the simulation, the winding and unwinding of the triple helix is possible, which leads to changes in the diameter of the Clps. To quantify variations, the diameter of the circle encompassed by the three CR atoms at position i of the three chains was calculated using 1 di ðtÞ ¼ 2ðai ðtÞ bi ðtÞ ci ðtÞÞpffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðai ðtÞ þ bi ðtÞ þ ci ðtÞÞ 1 1 pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðbi ðtÞ þ ci ðtÞ - ai ðtÞÞ ðai ðtÞ þ bi ðtÞ - ci ðtÞÞ 1 pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðci ðtÞ þ ai ðtÞ - bi ðtÞÞ

ð3Þ

where di(t) is the diameter at the ith position of the Clp at time t. ai, bi, and ci are distances between the CR atoms of each chain. The average diameter, Dia(t), of the triple helix at time t was calculated using DiaðtÞ ¼ Æd1 ðtÞ, d2 ðtÞ, :::, di ðtÞæ

Here, ~xi is the average position of atom i for the total number of frames T. 2.2.5. Hydrogen Bond Analysis. The occupancies of different types of hydrogen bonds were computed with maximal distance threshold for H-bond formation between the donor and acceptor non-hydrogen atoms of 3.5 Å. The following equation was used to classify the type of hydrogen bond between the given donor and acceptor atoms of the Clp: hydrogen bond typeðnÞi, jt dði, jÞ e 3:5 Å dði, W1 Þ e 3:5 Å and dðW1 , jÞ e 3:5 Å dði, W1 Þ e 3:5 Å, dðW1 , W2 Þ e 3:5 Å, and dðW2 , jÞ e 3:5 Å dði, W1 Þ e 3:5 Å, dðW1 , W2 Þ e 3:5 Å, dðW2 , W3 Þ e 3:5 Å, and dðW3 , jÞ e 3:5 Å otherwise

type value n was computed using hydrogen bond occði, jÞn P H - bond typeði, jÞt ¼ type n ¼ total no: of frames

ð4Þ

2.2.4. B-Factor. To understand the thermal fluctuation of individual residues and its effect on the overall structure, B-factors were computed for the backbone atoms (N, CR, C, and O) using 0 12 T 8 @1 X ðxi ðtj Þ - ~xi Þ2 A ð5Þ B-factor ¼ π 3 T tj ¼ 0

8 > > > 1, > > > 2, > > < 3, ¼ > 4, > > > > > > > : 0,

Figure 1. Definition of endocylic pseudorotation torsions ν0 to ν4 in imino acids.

ð6Þ

Here, d is the distance between the donor and the acceptor atoms, i, j, and W1-3 represent water donors or acceptors. If there is a direct hydrogen bond at time t between donor atom i and acceptor atom j, then the hydrogen bond type value is equal to 1. If there is a hydrogen bond network between donor i and acceptor j through a single water W1 at a time t, then the hydrogen bond type value is equal to 2. If there is a hydrogen bond network between donor i and acceptor j through two waters, namely, W1 and W2, at a time t, then the hydrogen bond type value is equal to 3. If there is a hydrogen bond network between donor i and acceptor j through three waters, namely, W1, W2, and W3 at a time t, then the hydrogen bond type value is equal to 4. If there is no interaction between i and j, then the hydrogen bond type value is equal to 0. The occupancy of a hydrogen bond between donor atom i and acceptor atom j of the

ð7Þ

2.2.6. Puckering Effect. The pseudo-rotation-phase angle P (0-360) of the five-membered rings (Pro and Hyp) was calculated as a function of time using eq 8.66 The endo cyclic torsions ν0 to ν4 are defined in Figure 1. tan P ¼

ðν4 þ ν1 Þ - ðν3 þ ν0 Þ 2ν4 ðsin 36 þ sin 72 Þ

ð8Þ

2.2.7. Radial Distribution Function. Consider a spherical shell of thickness δr at a distance r from a solute. The volume of the shell is given by 4 4 πðr þ δrÞ3 - πr 3 3 3 4 2 2 ¼ 4πr δr þ 4πr δr þ π δr 3 4πr 2 δr ð9Þ 3 If the number of particles per unit volume is F, then the total number in the shell is 4πFr2 δr, so the number of atoms in the volume element varies as r2. The radial distribution function (RDF), g(r), gives the probability of finding a solvent atom a distance r from another atom compared to the ideal solvent distribution, and therefore it is dimensionless. V

¼

3. RESULTS AND DISCUSSION 3.1. Effect of Guest AAs on the Overall Triple Helical Structure. Average values of Rg per residue, diameter, and rmsd

of backbone atoms for the model Clps are presented in Supporting Information Table 1. The radius of gyration per residue is about 0.85 Å for all the Clps, and it is in close agreement with previous results.54,55,65,66 The inner diameter computed from MD of Clps is about 5.7 Å, which is comparable with the crystal structure data.67-72 The mean rmsd values for host-guest peptides with the guest at XAA and YAA are 1.9 and 2.23 Å with reference to the initial structure. The properties of conventional hydrogen bonds formed between the amino group of Gly-N-H and the carboxyl group of XAA of the neighboring chain are listed in Supporting Information Table 2. The results show that the guest AAs do not affect the overall backbone hydrogen bonding pattern. Further, during the simulation, all Clps remain as triple helix and the substitution of AAs in the XAA and YAA positions 2596



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does not disrupt the overall triple helical structure of the host peptide, ((Gly-Pro-Hyp)3-Gly-XAA-YAA-(Gly-Pro-Hyp)3)3. The B-factors of all the backbone atoms for the model Clps are presented in Table 3. It can be seen that the thermal fluctuation Table 3. B-Factors of Backbone Atoms (N, Cr, C, O) of the Collagen-like Peptides (Å2) position XAA guest AA

a

ava

sda

position YAA av

sd

Pro/Hyp

48.6

47.1

48.6

47.1

Arg

48.3

42.8

49.3

48.2

Lys

43.3

40.1

39.1

34.4

Asp

52.7

35.1

67.0

64.9

Glu

48.9

41.2

43.4

45.1

His

63.3

60.3

90.3

58.4

Ser Thr

50.0 50.1

50.4 49.6

64.0 59.9

50.9 55.1

Asn

43.3

33.7

54.6

53.0

Gln

52.5

55.1

44.7

41.7

Cys

46.8

40.0

51.0

51.4

Gly

75.5

73.4

61.3

54.0

Ala

67.1

53.7

68.9

64.5

Val

71.8

58.7

58.8

66.1

Met Ile

82.6 60.4

51.1 62.8

46.3 48.9

42.6 42.1

Leu

55.8

53.8

68.8

56.8

Phe

100.6

70.7

54.9

52.7

Trp

83.8

47.3

45.4

42.7

Tyr

46.1

43.7

49.8

45.5

av: average, sd: standard deviation.

of the Clps varies with the guest AAs. In particular, a marked difference is noticeable in the aromatic AA substituted models. Typically, the guest peptide having Phe residue at the XAA position exhibits the largest B-factor value, which reiterates the earlier investigation by Bansal.73 Therefore, the presence of the guest AAs alters the structural stability and dynamics of the systems, and these differences are reflected in the melting temperatures. 3.2. Local Effect of Guest AAs on the Triple Helical Structure. 3.2.1. Diameter of the Triple Helix. It can be seen from the models that in XAA host-guest peptides, the guest AAs are present at the triad positions 9, 10, and 11. Similarly in YAA host-guest peptides, the guest AAs occupy the triad positions 10, 11, and 12. The average diameters of the triads 7-13 of the XAA and YAA host-guest peptides are presented in Figures 2 and 3. In particular, Glu at XAA decreases the diameter in all three substituted triads. A similar observation is also seen in the Leu-substituted Clp. The decrease in the diameter increases the compactness of the triple helix, and hence these AAs are observed frequently at XAA. On the other hand, change in the compactness of the triple helix at the substituted triads is minimal for all Clps with guest AAs except for those with Leu and Asn at YAA. These results reveal that propensity of an AA to adopt the triple helical conformation is position-dependent. 3.2.2. B-Factors of the Guest AAs. The B-factors of the backbone atoms of the guest AA are plotted in Figure 4. In comparison with Pro and Hyp, the presence of aromatic residues (Phe and Trp) and Met at XAA and His, Asp, and Leu at YAA amplifies fluctuation in the backbone atoms. Gly, Ala, and Val exhibit enhanced thermal fluctuations at XAA and YAA positions compared to the imino acids, indicating that smaller hydrophobic side chains in AAs induce more perturbations in the backbone. The presence of Arg and Lys in the

Figure 2. Average diameter of the triad numbers 7-13 of the collagen-like peptides with guest AA at Gly-XAA-Hyp. 2597



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Figure 3. Average diameter of the triad numbers 7-13 of collagen-like peptide with guest AA at Gly-Pro-YAA.

Figure 4. B-factor values of backbone atoms averaged over residues present in the substituted regions of collagen-like peptides.

triple helix tends to reduce the thermal fluctuation, which may be one of the reasons for the increase in the thermal stability (Table 1) of the collagen-like peptide with these residues. 3.2.3. φ and ψ Dihedral Angles in the Substituted Region. The replacement of Pro or Hyp by non-imino acids affects the backbone torsion angles. Incorporation of AAs at XAA is expected to affect the backbone dihedral angles, φ and ψ at the substituted region, i.e., the preceding Gly (XAA - 1), XAA, and succeeding Hyp (XAA þ 1). Similarly, substitution of AAs at YAA perturbs φ and ψ of the preceding Pro (YAA - 1), YAA, and succeeding Gly (YAA þ 1). The averages and standard

deviations of φ and ψ values of the above-mentioned residues upon substitution at XAA and YAA are given in Tables 4 and 5. It is evident from Table 4 that the presence of guest AAs at XAA induces conformational changes at XAA - 1 (ψ = 163-177) and XAA (ψ = 137-165) positions but not at XAA þ 1 (ψ = 147-150). Substantial changes are noticeable when aromatic AAs and Gly are present at the XAA position. It can be observed that significant changes take place only in the XAA - 1 position, and hence AAs affect the structural stability of triple helix toward the N-terminal direction with respect to the point of substitution. 2598



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Table 4. O and ψ Values (deg) of the Clps Substituted with Guest AA at XAA XAA - 1 (Gly) φ guest AA

a

ava

sda

av

sd

XAA þ 1 (Hyp)

XAA

ψ

φ av

Table 5. O and ψ Values (deg) of the Clps Substituted with Guest AA at YAA

ψ

φ

sd av sd

av

YAA - 1(Pro)

ψ sd

av

φ sd

guest AA

ava

ψ sda

av

YAA þ 1(Gly)

YAA φ

sd

av

ψ sd av

φ

sd

av

ψ sd

av

sd

Pro

-72 10 177 11 -71 10 160

9 -58

9

148

9

Hyp

-71 10 160

9 -58

9 148

9 -72 10 177 11

Arg

-70 10 166 13 -72 13 148 12 -59

9

150

10

Arg

-71 10 160

9 -63

9 147

8 -71 10 175 10

Lys Asp

-70 10 167 12 -72 12 150 11 -59 -69 10 165 12 -72 13 137 12 -59

9 9

150 147

10 10

Lys Asp

-70 10 157 9 -61 9 142 -68 11 153 12 -70 11 147

8 -69 9 170 10 9 -74 11 164 14

Glu

-70 10 164 12 -76 13 144 13 -60

9

148

10

Glu

-70 11 157 10 -63 10 141

8 -69

His

-69 10 168 12 -71 12 146 10 -60

9

150

10

His

-71 11 161 10 -65

9 150

9 -74 10 179 10

9 170 10

Asn

-70 10 163 12 -69 12 139 11 -59

9

149

10

Asn

-69 11 157 10 -62 10 140

9 -68 10 166 11

Gln

-70 10 165 12 -75 12 151 11 -60

9

150

10

Gln

-71 10 158

8 -68

Ser

-70 10 166 12 -73 13 149 11 -59

9

149

10

Ser

-70 11 158 10 -62 10 145

9 -71 10 172 12 8 -67

9 -63

9 144

9 -62 10 140

9 170

9

Thr

-70 10 162 12 -75 14 145 12 -60

9

150

10

Thr

-69 11 156

Cys Gly

-70 10 164 12 -73 13 148 13 -60 -72 11 171 14 -81 14 165 17 -60

9 10

149 149

10 10

Cys Gly

-69 11 157 10 -63 10 145 9 -71 10 173 11 -70 10 154 11 -64 10 147 11 -72 11 166 11

9 168 10

Ala

-71 10 169 13 -74 13 152 11 -60

9

149

10

Ala

-70 11 159 10 -63 10 147

9 -72 10 176 11

Val

-69 10 165 12 -77 13 154

8 -60

9

150

10

Val

-70 10 157

9 -61

9 142

7 -69

9 169

Met

-70 10 165 12 -75 13 148 11 -60

9

150

10

Met

-69 10 157

9 -62

9 142

8 -69

9 172 10

9

Ile

-69 10 165 12 -76 12 154

8 -61

9

150

10

Ile

-70 11 159

9 -60

9 142

8 -68

9 170

9

Leu

-71 10 162 13 -76 13 144 13 -60

9

149

10

Leu

-70 10 157

9 -57 10 135

8 -66

9 166

9

Phe

-71 10 166 14 -74 13 148 11 -60

9

150

10

Phe

-70 11 160 10 -62 10 146

9 -72 10 176 12

Trp Tyr

-70 10 169 15 -76 14 150 12 -61 -70 10 169 12 -73 12 148 10 -60

9 9

150 150

10 10

Trp Tyr

-72 10 162 10 -66 -70 11 160 10 -64

9 -75 10 181 12 9 -74 10 179 10


However the presence of non-imino acids at YAA induces conformational change at YAA - 1 (ψ = 153-162), YAA (ψ = 135-151), and YAA þ 1 (ψ = 164-181), and hence, changes are bidirectional with reference to the point of substitution. Thus the influence of the substitution of AA at YAA is significantly larger when compared to XAA. These structural variations are responsible for the noticeable changes in Tm values of guest AAs at YAA. Hence, the propensity of AAs to adopt the collagen conformation is significantly different for XAA and YAA positions. Therefore, the presence of Asp at YAA shows a significant decrease in φ and ψ, and the presence of Gly at XAA and YAA induces large conformational changes in the backbone.68,74 3.2.4. Effect of Guest AA on the Puckering of Neighboring Imino Acid. The role of puckering of imino acids on the structure and stability of collagen is well-documented.30,32,54-56,60,68,74 The puckering of the Hyp residue at XAA þ 1 of XAA host-guest Clps is given in Figure 5. Three puckering states, N exo, CR endo, and Cδ exo, are observed for Hyp. In particular, N exo and CR endo are more predominant in Gly-Pro-Hyp peptides. The presence of aromatic residues at XAA shifts the puckering of Hyp (XAA þ 1) from the N exo state to the Cγ exo or Cδ endo state. Conversely, the substitution of other AAs at the XAA position modifies the puckering of the Hyp (XAA þ 1) residue to the Cδ endo state. Figure 6 depicts the puckering of the Pro residue at YAA - 1 of YAA host-guest peptides. Pro exists in four puckering states as N endo, N exo, CR endo, and Cδ exo in the host peptides. The substitution of any AAs in YAA does not influence the puckering state of the neighboring Pro residue despite visible changes being observed in the ψ values of this residue. These results are consistent with

a

9 151 9 149


the previous X-ray structure and computationally predicted values.30,54-56,68,74 3.2.5. Additional Hydrogen Bonds. Crystallographic studies on Clps with charged residues, such as Arg and Lys, showed that the side chains of these residues interact only with the backbone carbonyl group of the nearby Gly residue.75,76 The side chains of polar amino acids, such as Asn, Thr, and Asp, were shown to make favorable interactions with the hydroxyl group of Hyp.68,74 Therefore, the presence of non-imino acids at XAA and YAA provides the possibility for new hydrogen bonds, in addition to the conventional ones in collagen. The simulations described here reveal that, in the Clps which contain guest AAs at XAA, the N-H of XAA forms a hydrogen bond with the hydroxyl group of Hyp of the neighboring chain. The new hydrogen bond is schematically presented in Figure 7. It is designated as an I 3 3 3 I-2 interchain hydrogen bond. It is found from the snapshots of MD simulations that this hydrogen bond is also mediated by water. Similarly, the water-mediated interchain hydrogen bond between XAA-N-H and Gly-CdO (I 3 3 3 Iþ2 interchain hydrogen bond) has also been observed. The occupancies of these hydrogen bonds and the number of water molecules mediating the hydrogen bond are given in Table 6. The occupancy and type of hydrogen bond between XAA-N-H and the hydroxyl group of Hyp differ with respect to the intrinsic nature of the residue at the XAA position. A single water mediated hydrogen bond is predominant in both cases. For example, the occupancy of the direct hydrogen bond (I 3 3 3 I-2) is around 36% for Leu at XAA, while, for Ile, it is 0.1%. If the Gly is substituted at the XAA position, a single water mediated hydrogen bond is 2599



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Figure 5. Effect of amino acids at the XAA position on the puckering of the XAA þ 1(Hyp) position.

observed between the XAA-N-H and the carboxyl group of the neighboring chain Gly (I 3 3 3 I-1, interchain). On the contrary, the N-H of AA at YAA fails to make any direct hydrogen bond with the neighboring residues; rather, it makes hydrogen bonds through water as YAA-N-H 3 3 3 YAA-CdO (designated as an I 3 3 3 I intrachain hydrogen bond), which is shown in Table 7. When compared to hydrogen bonds formed by N-H at XAA, the Clps substituted at YAA exhibit hydrogen bonds mediated by more than one water molecule. Therefore, hydrogen bonds formed by the N-H of non-imino acids may play a pivotal role in the origin of differences in enthalpy. However, the formation of additional hydrogen bonds is only possible when φ and ψ angles deviate from ideal values and, thereby, it affects the overall thermal stability. 3.2.6. Hydration Pattern. It has been shown in earlier studies that in the triple helical conformation, backbone atoms, such as the carboxyl group of Gly and YAA, and the amino group of XAA and YAA (except imino acids) interact directly with water molecules.74,77 If Hyp is present at YAA, then its hydroxyl group also participates in a hydrogen bonding interaction with water molecules.65,77 In addition, the side chains of AA can also interact with water molecules.65,74 The position-wise/sequence-dependent hydration pattern leads to a well-ordered

water network along the triple helical structure of collagen. To study the interaction of these atoms with water molecules, the RDF was computed. Integrated values of the first peak of RDF of water with respect to the backbone carboxyl oxygen of the positions 11, 12, and 13 of Clps are presented in Table 8. It can be observed that the hydration of the backbone carbonyl at the YAA position (∼2.0 water molecule) is higher than that of the same atom at the Gly position (∼1.4 water molecule), in accordance with the earlier experimental and modeling studies.54-56,77 From RDF analysis, it is clear that the hydration of the carboxyl group of the Gly is affected by the neighbor AA; in particular, it is noticeable in chains A and B. Substitution of AAs at the XAA position increases the hydration of the neighbor Gly (XAA - 1). On the other hand, substitution of the same at the YAA position marginally decreases the interaction of water molecules to the neighbor Gly (YAA þ 1). The integrated values of the first peak from the RDF of water with respect to the N-H of the guest AA at XAA and YAA are shown in Table 9. In the same table, the integrated values of the first peak of the RDF of water with respect to the O-H of Hyp (XAA þ 1) are also given. It can be observed that the N-H of AAs at XAA and YAA interact with at least one water molecule. This water interaction varies as A > B > C. The RDF 2600



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Figure 6. Effect of amino acids at the YAA position on the puckering of the YAA - 1 (Pro) position.

results are consistent with the hydrogen bond results for N-H of the guest AA. 3.2.7. Water Networks. The analysis of Clp hydration patterns from the MD simulations shows that it exhibits only marginal variations with respect to the properties of the AA present at the XAA and YAA positions. Even though the number of water molecules around the collagen peptides is similar, there is a possibility for differences in the organization of the water network. To understand the effect of water on the structure, hydrogen bonds mediated by water molecules were extracted from the trajectory. Two inter- and two intrachain hydrogen bonds mediated by water molecules per triplet were observed in the crystal structures of Clps;68,74,77 they are (a) CdO of Gly at the position I of one chain and CdO of YAA at the position I-1 of the neighbor chain named as I 3 3 3 I-1 interchain hydrogen bond, (b) CdO of Gly at the position I of one chain and O-H of Hyp at the position I-4 of the neighbor chain named as I 3 3 3 I-4 interchain hydrogen bond, (c) CdO of Gly at position I and CdO of YAA at position I-1 of the same chain named as the I 3 3 3 I-1 intrachain hydrogen bond, and (d) CdO of Gly at position I and O-H of Hyp at position Iþ2 of the same chain named as the I 3 3 3 Iþ2 intrachain hydrogen bond. It has been noted that the second and fourth categories

Figure 7. Snapshots of a section of the simulated collagen-like peptide triple helix showing the new direct hydrogen bond (left) and the previously established water-mediated hydrogen bond (right) (lengths are in angstroms). The hydrogen bond is formed between the Hyp OH and the Ala (XAA) N-H on the adjacent chain (the side chains of other residues are omitted for clarity). Dihedral angles (deg) are given for the Ala for the respective hydrogen-bonding patterns.

are possible only when Hyp is present. Occupancies of interchain hydrogen bonds in various Clps and differences in their occupancies with respect to imino acids for various AAs are 2601



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Table 6. Occupancies (%) of Additional Interchain Hydrogen Bond Formed by the N-H of AAs at XAA XAA-N-H 3 3 3 Hyp-O-D (I 3 3 3 I-2)

XAA-N-H 3 3 3 Gly-CdO (I 3 3 3 Iþ2)

water-mediated

water-mediated

direct

1W

2W

3W

1W

2W

3W

no interaction

Pro

-

-

-

-

-

Arg

22.9

61.7

5.7

0.0

Lys

5.5

76.5

6.3

0.0

9.7

65.7

5.0

2.3

26.6

11.7

82.5

3.6

1.4

Asp

11.8

70.8

4.5

11.0

0.0

12.9

74.7

3.0

2.2

Glu

22.0

60.8

19.3

4.0

2.3

11.0

60.7

5.4

2.1

His

4.9

31.1

75.5

7.3

0.0

12.3

81.4

3.6

1.9

Ser

11.0

14.5

63.7

7.6

0.0

14.3

62.9

11.0

4.7

20.5

Thr Asn

25.0 20.7

52.6 62.4

4.9 6.7

0.0 0.0

17.5 10.3

46.0 67.1

10.8 5.2

3.5 3.8

37.7 22.6

Gln

6.7

59.3

8.8

0.0

25.3

69.9

4.9

2.3

21.6

Cys

15.3

62.5

7.0

0.0

15.3

65.3

6.4

2.8

23.9

Gly

21.2

50.1

14.7

0.0

14.0

48.7

17.6

9.8

21.3

Ala

6.4

71.1

9.1

0.0

13.4

78.6

6.2

2.6

11.7

Val

0.5

66.9

9.1

0.0

23.4

79.7

2.0

1.0

16.8

Met

22.8

59.5

4.3

0.0

13.5

60.8

5.0

2.1

31.5

Ile Leu

0.1 36.3

72.5 48.7

7.5 3.2

0.0 0.0

19.9 11.8

84.2 46.4

1.3 5.0

0.5 2.1

13.5 44.7

Phe

9.9

68.5

6.6

0.0

15.0

71.9

5.7

2.1

19.4

Trp

22.7

59.9

5.0

0.0

12.5

62.6

5.3

2.4

29.2

Tyr

4.2

75.4

7.2

0.0

13.2

82.9

3.0

2.0

11.2

guest AA at XAA

Table 7. Occupancies (%) of YAA-N-H 3 3 3 YAA-CdO (I 3 3 3 I) Intrachain Hydrogen Bond occupancy water-mediated guest AA at YAA

1W

Hyp

-

-

-

-

Arg

0.00

10.21

16.52

73.28

Lys

0.00

20.02

25.85

54.14

Asp Glu

0.00 0.00

24.04 30.65

10.59 26.90

65.38 42.45

His

0.00

10.16

9.94

79.90

Ser

0.00

23.09

23.85

53.06

Thr

0.00

22.37

26.77

50.86

Asn

0.00

23.06

26.31

50.63

Gln

0.00

22.56

28.00

49.44

Cys

0.00

19.68

24.33

55.99

Gly Ala

0.00 0.00

27.35 22.94

29.90 26.15

42.75 50.92

Val

0.00

4.64

12.52

82.84

Met

0.00

23.00

30.37

46.63

Ile

0.00

11.20

13.98

74.82

Leu

0.00

16.82

28.15

55.04

Phe

0.00

14.41

11.68

73.91

Trp

0.00

4.81

7.16

88.03

Tyr

0.00

13.12

8.75

78.13

2W

3W

no interaction

listed in Table 10. The same information for the intrachain hydrogen bond is depicted in Table 11.

no interaction

I 3 3 3 I-1 interchain water-mediated hydrogen bond is not favorable in the host sequence Gly-Pro-Hyp (only present for 18.8% of the trajectory). Substitution of AAs at X AA leads to the formation of this interchain hydrogen bond mediated by two or three water molecules. In particular, Asp at X AA favors the formation of this water-mediated interaction. Substitution of the same AAs at YAA does not favor this interaction. As a consequence, the stability of the same peptide decreases, as evident from the melting temperature measurements.44 The interchain hydrogen bond mediated by water molecules I 3 3 3 I-4 is observed in various crystal structures and has been attributed to the hydroxylation of Pro at YAA. Three or two water molecules are essential for this interchain hydrogen bond. Substitution of AAs at the XAA and YAA positions disrupts these interactions which lead to a decrease in the stability of the triple helix when compared to the host peptide. The intrachain hydrogen bond I 3 3 3 I-1 is mediated by two or three water molecules. The MD simulation reveals that the formation of this interaction with a single water molecule is rare. The presence of other AAs at XAA tends to slightly destabilize the formation of this intrachain water-mediated hydrogen bond. However, other AAs at YAA tend to relatively stabilize the interaction. The I 3 3 3 Iþ2 intrachain water-mediated hydrogen bond is mostly formed through 1 or 2 water molecules. Substitution of AAs at XAA and YAA in general favors this hydrogen bond. However, substitutions of Arg, His, Gly, and Tyr at YAA do not stabilize this water-mediated hydrogen bond. Since the triple helical structure has an inside out nature, the side chains of AAs at XAA and YAA positions are exposed to solvent. Thus, substitution of these residues influences the water-mediated hydrogen bonds present in the triple helical structure. 2602



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Table 8. Integrated Values (Given in Number of Molecules (Water)) of the First Peak from the Radial Distribution Function Calculated between Water and CdO of AAs guest AA

11th position Gly-CdO

position chain

XAA A

B

13th position YAA-CdO

YAA C

A

XAA

B

C

A

B

14th position Gly-CdO

YAA C

A

XAA

B

C

A

B

YAA C

A

B

C

Pro/Hyp

1.43

1.42

1.39

1.43

1.42

1.39

1.98

2.09

1.97

1.98

2.09

1.97

1.42

1.40

1.44

1.42

1.40

1.44

Arg

1.96

1.81

1.46

1.49

1.38

1.42

2.02

2.27

2.11

2.26

0.74

1.65

1.42

1.52

1.40

1.36

0.98

1.28

Lys Asp

1.96 1.90

2.04 1.80

1.48 1.53

1.39 1.50

1.47 1.46

1.40 1.39

1.98 2.47

2.06 2.59

2.02 2.53

2.28 2.46

1.49 2.58

1.44 2.69

1.37 1.48

1.40 1.43

1.41 1.46

1.29 1.67

1.26 1.77

1.30 1.59

Glu

1.82

1.89

1.72

1.47

1.41

1.42

2.31

2.26

2.22

2.39

2.36

2.35

1.49

1.45

1.45

1.50

1.55

1.56

His

1.98

1.86

1.37

1.47

1.44

1.41

1.86

1.85

1.84

1.97

1.87

1.83

1.54

1.60

1.59

1.26

1.32

1.26

Ser

1.80

1.84

1.61

1.46

1.42

1.40

2.14

2.25

2.28

2.27

2.33

2.30

1.45

1.48

1.46

1.41

1.45

1.40

Thr

1.78

1.81

1.63

1.46

1.45

1.45

2.09

2.07

2.29

2.31

2.07

2.04

1.52

1.43

1.45

1.32

1.29

1.34

Asn

1.78

1.70

1.48

1.46

1.45

1.41

2.17

1.97

2.06

2.19

2.46

2.42

1.48

1.57

1.51

1.28

1.43

1.49

Gln

1.92

1.92

1.57

1.45

1.41

1.38

2.01

2.09

2.10

2.34

1.88

1.40

1.40

1.44

1.42

1.37

1.32

1.14

Cys Gly

1.73 1.92

1.90 1.90

1.54 1.57

1.41 1.46

1.43 1.43

1.37 1.41

2.17 2.32

2.18 2.42

2.11 2.24

2.21 2.43

2.23 2.82

2.04 2.77

1.50 1.58

1.44 1.49

1.42 1.47

1.37 1.69

1.56 1.71

1.34 1.66

Ala

1.98

1.99

1.46

1.42

1.44

1.46

2.09

2.15

2.09

2.30

2.28

2.21

1.41

1.45

1.40

1.51

1.45

1.46

Val

2.02

2.06

1.46

1.44

1.43

1.39

2.00

1.86

2.08

1.84

1.68

1.66

1.37

1.46

1.41

1.29

1.32

1.33

Met

1.73

1.89

1.52

1.42

1.41

1.41

2.10

2.22

2.10

2.32

1.78

1.71

1.52

1.37

1.34

1.31

1.27

1.29

Ile

2.01

1.91

1.50

1.44

1.46

1.48

1.97

2.06

2.02

2.13

1.70

1.44

1.44

1.40

1.44

1.33

1.30

1.27

Leu

1.74

1.67

1.55

1.40

1.47

1.40

2.13

2.31

2.10

2.18

1.85

1.90

1.54

1.44

1.40

1.27

1.26

1.29

Phe

2.06

1.93

1.43

1.47

1.44

1.37

1.93

1.97

1.75

2.04

1.78

1.74

1.48

1.61

1.58

1.29

1.27

1.22

Trp Tyr

1.93 1.97

1.91 1.94

1.55 1.46

1.42 1.44

1.46 1.42

1.46 1.41

2.02 1.66

1.73 1.94

1.83 1.66

1.92 2.06

1.54 1.85

1.37 1.76

1.52 1.58

1.69 1.59

1.49 1.62

1.28 1.27

1.23 1.21

1.19 1.76

Table 9. Integrated Values (Given in Number of Molecules (Water)) of the First Peak from the Radial Distribution Function Computed between Water and N-H of AAs at XAA and YAA and O-H of Hyp guest AA

12th position XAA-N-H

13th position Hyp-O-H

13th position YAA-N-H

position

XAA

XAA

YAA

chain

A

B

C

A

B

C

A

B

C

Pro/Hyp

-

-

-

3.36

3.27

2.76

-

-

-

Arg

1.06

1.43

1.00

3.21

2.66

2.75

1.46

1.21

1.27

Lys

1.64

1.22

1.16

2.48

2.53

2.81

1.43

1.48

1.33

Asp

1.49

0.45

1.25

2.95

3.03

2.76

1.20

1.27

1.26

Glu

1.16

0.95

1.05

3.17

3.21

2.83

1.50

1.50

1.46

His Ser

1.37 1.30

1.49 1.32

1.46 1.15

3.08 3.31

3.03 3.09

2.78 2.80

1.13 1.50

1.13 1.50

1.14 1.47

Thr

1.42

0.93

0.78

2.93

3.04

2.80

1.51

1.45

1.45

Asn

1.50

1.33

1.17

2.86

2.75

2.84

1.48

1.48

1.42

Gln

1.44

1.41

0.99

2.83

3.10

2.78

1.43

1.45

1.42

Cys

1.33

1.04

1.40

3.13

3.19

2.86

1.40

1.34

1.25

Gly

1.44

1.74

1.64

3.24

3.20

2.82

1.69

1.72

1.74

Ala

1.45

1.49

1.34

2.88

3.33

2.76

1.43

1.41

1.41

Val Met

1.19 1.20

1.13 0.81

1.05 0.97

3.31 3.04

3.23 2.90

2.71 2.74

1.16 1.47

1.18 1.47

1.19 1.43

Ile

1.19

1.21

1.01

3.11

3.18

2.78

1.11

1.15

1.15

Leu

1.32

0.58

0.58

2.82

3.12

2.75

1.40

1.41

1.40

Phe

1.23

1.30

1.42

3.72

3.52

2.88

1.06

1.06

1.07

Trp

0.97

1.44

1.03

3.23

2.78

2.76

1.05

1.08

0.97

Tyr

1.56

1.34

1.48

3.14

3.23

2.75

1.09

1.08

1.07

2603



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Table 10. Occupancy (%) of Interchain Water-Mediated Hydrogen Bond of Collagen-like Peptidesa position

a

XAA

YAA

guest AA

1W

2W

3W

no interaction

Pro

0.6

3.8

14.4

81.2

Arg

1.4

13.2

27.4

58.0

difference

1W

Gly-CdO 3 3 3 YAA-CdO (I 3 3 3 I-1) 0.0 0.6 23.3

0.1

2W

3W

no interaction

difference

3.8

14.4

81.2

0.0

1.0

4.9

94.0

-12.8 -11.4

Lys

2.5

10.9

25.6

61.0

20.3

0.0

1.1

6.3

92.6

Asp

2.6

19.2

35.5

42.8

38.5

0.8

11.3

16.0

71.9

9.4

Glu

2.0

16.3

30.1

51.5

29.7

0.0

1.0

7.1

91.9

-10.7 -9.5

His

2.3

8.4

30.7

58.6

22.6

0.1

1.7

7.4

90.8

Ser

1.9

13.7

29.7

54.7

26.6

0.3

2.8

16.1

80.9

0.4

Thr Asn

3.5 1.4

17.2 12.1

27.8 26.5

51.5 60.0

29.7 21.3

0.0 0.2

0.6 1.4

6.2 6.9

93.2 91.4

-12.0 -10.2

Gln

1.4

8.8

26.8

62.9

18.3

0.0

1.0

6.4

92.5

-11.3

Cys

2.1

13.9

30.3

53.6

27.6

0.1

2.0

13.4

84.5

-3.2

Gly

0.7

7.5

24.4

67.4

13.8

1.4

18.1

25.3

55.2

26.1

Ala

0.9

6.6

25.7

66.8

14.4

0.0

1.9

14.1

84.0

-2.7

Val

1.9

10.9

25.3

61.9

19.3

0.0

1.0

6.0

92.9

-11.7

Met

1.5

14.7

30.5

53.3

27.9

0.1

0.6

5.3

94.0

-12.8

Ile Leu

0.8 5.0

9.4 21.7

25.7 26.8

64.0 46.4

17.2 34.8

0.0 0.3

0.7 1.3

5.2 6.3

94.0 92.2

-12.8 -10.9 -10.6

Phe

1.8

9.9

29.3

58.9

22.3

0.0

1.2

6.9

91.9

Trp

3.1

11.7

29.1

56.0

25.2

0.3

2.9

10.2

86.6

-5.4

Tyr

1.6

13.7

32.6

52.1

29.1

0.1

1.2

7.1

91.6

-10.4

Pro

0.1

7.6

26.6

65.7

Arg

0.0

1.7

9.4

Lys Asp

0.0 0.0

1.4 2.3

11.2 9.3

Gly-CdO 3 3 3 Hyp-O-H (I 3 3 3 I-4) 0.0 0.1

7.6

26.6

65.7

0.0

88.8

-23.1

0.1

4.7

20.1

75.2

-9.5

87.3 88.4

-21.6 -22.7

0.0 0.0

5.1 4.9

18.5 18.9

76.3 76.1

-10.6 -10.4

Glu

0.0

1.3

7.9

90.8

-25.0

0.0

4.7

20.1

75.2

-9.4

His

0.0

2.4

13.3

84.3

-18.5

0.0

4.9

21.6

73.4

-7.7

Ser Thr

0.0 0.0

1.8 0.7

9.8 5.3

88.4 93.9

-22.6 -28.2

0.0 0.1

4.5 4.9

18.3 20.8

77.2 74.2

-11.5 -8.4

Asn

0.0

1.9

9.0

89.2

-23.5

0.0

4.9

19.1

76.0

-10.2

Gln

0.0

1.6

10.3

88.1

-22.4

0.0

3.6

16.7

79.7

-13.9

Cys Gly

0.0 0.1

1.4 4.1

9.2 19.1

89.4 76.7

-23.7 -11.0

0.0 0.0

4.3 3.5

18.2 15.2

77.5 81.3

-11.8 -15.5

Ala

0.0

3.1

15.5

81.4

-15.6

0.1

6.1

23.0

72.0

-6.3

Val

0.0

1.4

10.4

88.2

-22.5

0.1

5.7

21.5

72.8

-7.0

Met

0.0

1.3

8.1

90.6

-24.9

0.0

5.4

20.3

74.2

-8.5

Ile

0.0

1.7

10.2

88.1

-22.3

0.1

7.6

25.3

67.0

-1.3

Leu

0.0

0.4

4.2

95.5

-29.7

0.1

5.5

20.3

74.1

-8.4

Phe

0.0

2.3

11.9

85.7

-20.0

0.1

5.4

23.4

71.1

-5.3

Trp Tyr

0.0 0.0

1.5 1.8

8.9 11.0

89.6 87.2

-23.9 -21.5

0.0 0.1

4.0 5.0

16.8 21.7

79.2 73.1

-13.5 -7.4

iW in the column headings represent the occupancy of the i number of water molecules that mediates the hydrogen bond.

4. CONCLUSIONS Molecular dynamics simulations of 39 model Clps reveal the structural basis for variations in the propensity of AAs to adopt the collagen conformation. The propensity of various AAs to adopt collagen-like conformations depends primarily on the φ and ψ angles. In addition to these geometric parameters, the orientation of the side chain with respect to

the backbone is important. Some of the AAs (Gly, Ile at YAA) exhibit significant deviation from the ideal values of φ and ψ for collagen-like conformation in the Ramachandran plot. Hence, their percentage occurrence in collagen is lower. The φ and ψ values for various AAs and their respective positions in the Gly-XAA-YAA repeat dictate the formation of hydrogen bonds with the neighboring chain as well as 2604



ARTICLE

Table 11. Occupancy (%) of Interchain Water-Mediated Hydrogen Bond of Collagen-like Peptidesa position

a

XAA

YAA

guest AA

1W

2W

3W

no interaction

difference

Pro

3.5

49.3

25.0

22.2

Arg

4.7

43.0

24.1

28.2

-6.1

1W

Gly-CdO 3 3 3 YAA-CdO (I 3 3 3 I-1) 0.0 3.5 2.2

2W

3W

no interaction

difference

49.3

25.0

22.2

0.0

49.5

22.2

26.1

-4.0

Lys

3.9

38.7

24.6

32.9

-10.7

2.3

52.8

24.2

20.8

1.4

Asp

4.1

41.5

26.1

28.3

-6.2

6.6

48.5

23.7

21.1

1.0 7.3

Glu

4.4

47.1

22.7

25.7

-3.6

2.4

56.1

26.7

14.8

His

3.6

47.5

22.3

26.7

-4.5

2.8

53.4

25.1

18.7

3.4

Ser

4.4

40.0

23.8

31.8

-9.6

2.7

55.4

23.9

18.1

4.1

Thr Asn

4.1 2.7

43.7 54.4

24.1 22.2

28.1 20.6

-5.9 1.5

2.2 2.1

57.3 56.9

24.3 24.0

16.2 17.0

5.9 5.2 2.3

Gln

3.8

45.0

22.8

28.4

-6.2

2.1

54.5

23.5

19.9

Cys

4.0

44.1

22.9

28.9

-6.8

2.7

54.0

25.1

18.2

4.0

Gly

3.8

49.4

20.6

26.2

-4.1

4.5

49.9

24.4

21.1

1.0

Ala

3.8

49.8

22.6

23.8

-1.6

2.8

57.8

23.5

15.9

6.3

Val

4.5

46.8

21.9

26.8

-4.7

2.3

49.0

28.7

20.0

2.2

Met

4.5

42.7

22.1

30.7

-8.5

2.1

60.5

22.6

14.9

7.3

Ile Leu

4.0 4.3

42.5 44.4

25.4 23.3

28.1 28.0

-5.9 -5.8

2.1 1.8

56.2 61.0

25.3 23.0

16.4 14.1

5.8 8.0

Phe

3.8

48.9

23.5

23.8

-1.6

2.7

57.4

23.0

16.9

5.2

Trp

4.6

44.8

23.1

27.5

-5.3

2.7

51.6

25.0

20.6

1.6

Tyr

4.9

46.2

22.7

26.3

-4.1

2.5

55.4

23.6

18.5

3.6

Pro

10.1

45.2

26.5

Gly-CdO 3 3 3 Hyp-O-H (I 3 3 3 Iþ2) 18.2 0.0 10.1

45.2

26.5

18.2

0.0

Arg

44.0

29.9

15.5

10.0

8.2

10.6

44.5

20.2

24.7

-6.5

Lys Asp

47.0 57.1

27.8 21.3

14.6 13.1

10.5 8.0

7.7 10.2

14.9 11.9

47.1 42.1

21.3 32.1

16.7 14.0

1.5 4.3

Glu

47.9

27.0

15.8

9.4

8.8

15.8

45.1

24.7

14.4

3.8

His

50.5

26.4

13.8

9.1

9.1

12.9

42.7

23.8

20.6

-2.4

Ser

42.3

30.4

17.2

9.8

8.4

11.6

42.5

27.2

18.6

-0.4

Thr

38.7

31.3

17.8

11.8

6.4

22.1

42.3

20.7

14.9

3.3

Asn

49.9

25.7

14.1

9.8

8.4

18.6

41.7

23.7

16.0

2.2

Gln

39.8

29.3

17.4

13.3

5.0

10.4

48.9

23.5

17.2

1.0

Cys Gly

42.6 27.3

29.2 34.2

16.8 24.5

11.1 14.0

7.1 4.3

12.5 12.6

42.7 35.4

26.5 31.7

18.3 20.3

-0.1 -2.1

Ala Val

43.8 45.0

28.3 28.8

17.3 16.0

10.5 10.2

7.7 8.0

9.9 27.5

41.8 39.8

29.9 18.7

18.5 14.0

-0.3 4.2

Met

43.3

28.8

16.7

10.7

7.5

18.8

46.3

20.7

14.3

3.9

Ile

45.4

26.5

17.4

10.7

7.5

29.9

37.9

18.9

13.4

4.8

Leu

43.7

29.2

15.8

10.3

8.0

32.2

39.6

17.3

10.9

7.3

Phe

48.8

26.2

14.7

10.1

8.1

13.5

45.2

22.4

18.9

-0.7

Trp Tyr

46.2 47.6

27.0 26.7

15.8 16.5

10.2 9.1

8.0 9.1

9.9 14.3

40.1 44.1

29.7 21.9

20.2 19.7

-2.0 -1.5

iW in the column headings represent occupancy of the i number of water molecules that mediate the hydrogen bond.

water-mediated hydrogen bonding. Analysis of the hydrogen bonding properties showed that there are no noticeable differences in the conventional hydrogen bonding interaction in collagen upon substitution of various AAs in the XAA and YAA positions in the host peptide sequence. However, the presence of some AAs leads to the formation of a new direct hydrogen bond between the N-H at XAA and the O-H

of Hyp. Further, depending on the AA type, hydrogen bonding between these groups can be mediated by water molecules. In addition to this hydrogen bonding, the nature of the side chains of guest AA residue also influences the local hydration pattern and puckering of the neighbor Hyp in the structure. The abovementioned factors determine the structural propensity of each AA on the collagen. 2605



’ ASSOCIATED CONTENT

bS

Supporting Information. Tables listing structural and conventional hydrogen bond Gly-N-H 3 3 3 XAA-O properties of collagen-like peptides. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT We acknowledge the Department of Science and Technology (DST), the Department of Biotechnology (DBT), the Council of Scientific and Industrial Research (CSIR), India, the German Academic Exchange Service (DAAD), Germany, and the Klaus Tschira Foundation for financial support. S.S.R. thanks CSIR, India for providing the Senior Research Fellowship and the DAAD for a predoctoral Sandwich Scholarship. ’ REFERENCES (1) Brodskyk, B.; Persikov, A. V. Molecular structure of the collagen triple helix. Fibrous Proteins: Coiled-Coils, Collagen and Elastomers 2005, 70, 301. (2) Hulmes, D. J. S. The collagen superfamily—Diverse structures and assemblies. Essays Biochem. 1992, 27, 49. (3) Scordino, A.; Grasso, R.; Gulino, M.; Lanzano, L.; Musumeci, F.; Privitera, G.; Tedesco, M.; Triglia, A.; Brizhik, L. Hydration Effects on Photophysical Properties of Collagen. In Self-Organization of Molecular Systems: From Molecules and Clusters to Nanotubes and Proteins; Russo, N., Antonchenko, V. Y., Kryachko, E. S., Eds.; Springer: Berlin, 2009; Vol. 14, 359. (4) Exposito, J. Y.; Valcourt, U.; Cluzel, C.; Lethias, C. Int. J. Mol. Sci. 2010, 11, 407. (5) Hattori, S. Sen-i Gakkaishi 2009, 65, 453. (6) Fullerton, G. D.; Rahal, A. J. Magn. Reson. Imaging 2007, 25, 345. (7) Bravo, J. F. Rev. Med. Chile 2009, 137, 1488. (8) Bodian, D. L.; Madhan, B.; Brodsky, B.; Klein, T. E. Biochemistry 2008, 47, 5424. (9) Persikov, A. V.; Pillitteri, R. J.; Amin, P.; Schwarze, U.; Byers, P. H.; Brodsky, B. Hum. Mutat. 2004, 24, 330. (10) Kahan, V.; Andersen, M. L.; Tomimori, J.; Tufik, S. Brain Behavior and Immunity 2009, 23, 1089. (11) Brodsky, B.; Baum, J. Nature 2008, 453, 998. (12) Baum, J.; Brodsky, B. FASEB J. 1998, 12, 116. (13) Li, Y. J.; Brodsky, B.; Baum, J. J. Biol. Chem. 2009, 284, 20660. (14) Liu, X. Y.; Kim, S.; Dai, Q. H.; Brodsky, B.; Baum, J. Biochemistry 1998, 37, 15528. (15) Battineni, M. L.; Yang, W.; Liu, X. Y.; Baum, J.; Brodsky, B. Matrix Biol. 1996, 15, 174. (16) Ramachandran, G. N.; Kartha, G. Nature 1955, 176, 593. (17) Rich, A.; Crick, F. H. C. Nature 1955, 176, 915. (18) Okuyama, K.; Okuyama, K.; Arnott, S.; Takayanagi, M.; Kakudo, M. J. Mol. Biol. 1981, 152, 427. (19) Leikin, S.; Parsegian, V. A.; Yang, W. H.; Walrafen, G. E. Proc. Natl.Acad. Sci. U.S.A. 1997, 94, 11312. (20) Bhatnagar, R. S.; Gough, C. A.; Qian, J. J.; Shattuck, M. B. Proc.-Indian Acad. Sci., Chem. Sci. 1999, 111, 301. (21) Melacini, G.; Bonvin, A.; Goodman, M.; Boelens, R.; Kaptein, R. J. Mol. Biol. 2000, 300, 1041. (22) Xu, Y. J.; Persikov, A.; Jordan, J. A.; Brodsky, B. Biophys. J. 2000, 78, 2504. (23) Xu, Y. J.; Bhate, M.; Brodsky, B. Biochemistry 2002, 41, 8143.

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Structural Basis for the Varying Propensities of Different Amino Acids

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