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CHAPTER 1

INTRODUCTION

1.1 COLLAGEN: THE MAJOR CONNECTIVE TISSUE

PROTEIN

Collagen, the major constituent of the extracellular matrix, is the

most abundant structural protein in all higher organisms (Di Lullo et al 2002).

It is mostly found in fibrous tissues such as tendon, ligament and skin in the

form of elongated fibrils and is also abundant in cornea, cartilage, bone, blood

vessels, the gut, and intervertebral disc. Till date, 29 types of collagen have

been identified. These are

forty two different genes (Heino 2007). Type I, II, and III are most abundant

and provides the scaffolding and guide cells to migrate, proliferate and

differentiate (Gullberg et al 1992; Tuckwell et al 1994; Farndale et al 2008).

According to their supramolecular morphology, collagen is usually classified

into five groups: (i) fibril forming, (ii) fibril associated containing interrupted

triple helices, (iii) beaded filament, (iv) anchoring fibril, and (v) network

forming and transmembrane collagens (Ricard-Blum et al 2005; Khoshnoodi

et al 2006).

Collagen is widely used in biomedical and industrial applications

(Stenzel et al 1974; Oryan 1995; Ramshaw et al 1996; Olsen et al 2003;

Fields 2010; Koide 2005). It has been employed as a core material for 3D

scaffolding in tissue engineering and cell therapy as well as collagen sheets

for wound healing. Collagen is used as a drug delivery material and in

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cosmetic industries to prevent ageing (Olsen et al 2003). In leather industry,

the degradation of hides and skin by bacterial collagenase is prevented by the

interaction of collagen with a variety of tanning agents (Hand et al 2000;

Sreeram et al 2000a, 2000b, 2004; Thanikaivelan et al 2000; Fathima et al

2003a, 2003b, 2004, 2005, 2006a, 2006b, 2006c, 2007, 2009, 2010; Usha et al

2000, 2009, 2010, 2012; Gayathiri et al 2001; Tang et al 2003; Madhan et al

2001, 2003a, 2003b, 2003c, 2005, 2007; Jones et al 2007; Kanth et al 2006;

Krishnamoorthy et al 2008; Covington 2009; Sehgal et al 2009; Wu et al

2009; Chen et al 2010; Gong et al 2010; Mitra et al 2011a, 2011b).

1.2 COLLAGEN STRUCTURE AND STABILITY

1.2.1 Collagen Structure

Ramachandran et al (1955) proposed a three dimensional structure

for collagen, which is also known as Madras model by using fibre diffraction

pattern of kangaroo tail tendon. Later in the same year, Rich and Crick (1955)

refined the triple helical structure with more stringent stereo chemical criteria.

According to both the models, three polypeptide chains forms a coiled coil

conformation. A unique tertiary structure, triple helix is the most striking

feature of the collagen molecule. The three identical or non-identical

polypeptide chains form the triple helical structure of collagen. Each chain is

of about 1000 amino acids or more in length in some collagen types. The

three polypeptide chains are super coiled in a left handed manner around a

common axis, with a staggering of one residue between adjacent chains

leading to a single extended right-handed triple helical conformation. Only

glycine can be accommodated in the interior of the triple helix without chain

distortion due to the close packing of three chains about a common axis,

which leads to a steric constraint on every third residue. Non helical terminals

of the triple helix are called as N, C-telopeptides. These telopeptides play an

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important role in micro-fibril and fibril formation. Schematic representation

of triple helical structure is presented in Figure 1.1.

Figure 1.1 Triple helical structure shown for (Gly-Pro-Hyp)n in which

Gly, Pro and Hyp are designated as 1(Gly), 2(XAA) and 3(YAA)

respectively. (a) left-handed polyproline-II like helices, (b) right-handed

supercoil, (c) Top view in the direction of the helical axis (Courtesy:

Brinckmann et al 2005)

1.2.2 Factors Influencing Stabilization of Triple Helix in Collagen

1.2.2.1 Stability of various types of collagen

Collagen shows different thermal stability (Tm) in each organism

and tissue (Kurz et al 2004; Persikov et al 2004; Yang et al 2004; Cavalcante

et al 2005; Trebacz et al 2005; Calderon et al 2010). This temperature

strongly correlates with body temperature of the species (Rose et al 1988). In

addition, an experiment on the melting temperature of type II collagen reveals

the variation in the thermal stability within the triple helical domain

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(Steplewski et al 2004). Besides, mutations in the thermo labile domains

destabilize the entire molecule lower than that of thermo stable domains.

1.2.2.2 Hydrogen bonds

The tightly packed right handed triple helix is stabilized through

hydrogen bonding network and van der Waals interactions. The nature of

hydrogen bonding pattern in triple helix has been proposed by Ramachandran

and Kartha (1955) and Rich and Crick (1955). The hydrogen bonding pattern

proposed by Ramachandran and Crick are presented schematically in Figure

1.2.

Figure 1.2 Schematic representation of hydrogen bonding pattern

present in the proposed triple helix structure (Courtesy: Vijayan et al

1999)

According to Ramachandran model (1955), triple helical structure

is stabilized by the formation of two hydrogen bonds per G-XAA-YAA triplet.

This model postulates two requirements: (i) glycine should be present at every

third position, (ii) imino acids preferably accommodates the Y position. Triple

helix may be stabilized by (i) direct hydrogen bond formed by glycine amino

groups, and (ii) hydrogen bond formed via water molecules with hydroxyl

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group of the amino acid residue such as Hyp, Ser, and Thr. The same has been

supported by Broadsky and coworkers with 1.9 Å high resolution crystal

structure (Bella et al 1994). Rich and Crick proposed a model with more

stringent stereo chemical criteria. This model retains all the essential features

of Ramachandran coiled-coil triple helical model except the number of

hydrogen bonds. It contains one hydrogen bond per G-XAA-YAA tri peptide

unit, the amide group of the Gly forms a hydrogen bond with the carbonyl

group of the residue in the X position in the adjacent chain of the triple helix,

(Gly)N-H…O=C(XAA). This model has been supported by several authors

with 1.7 Å high resolution crystal structure (Okuyama et al 2006, 2008;

Kramer et al 1998).

1.2.2.3 Water network

Crystal structure of collagen like peptides (CLPs) helps us to

understand the role of water on the triple helical structure (Bella et al 1995).

An extensive hydration network satisfies the peptide backbone groups, which

have been shown in Figure 1.3.

Figure 1.3 Schematic representations of types of hydrogen bonding

patterns found in the triple-helix (Courtesy: Bella et al 1995)

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1.2.2.4 Hydroxylation of Pro residue in YAA position

The imino acid content of collagen is about 25%, with at least half

in the form of hydroxyproline. The hydroxylation of Pro residues in collagen

dramatically increases the thermal stability of triple helices. Stabilization

occurs if Hyp is present in the YAA position with 4R configuration of

hydroxyl group but not in the XAA position, nor in 4S configuration (Bann et

al 2000; Mizuno et al 2004; Barth et al 2003, 2004; Doi et al 2003, 2005;

Hodges et al 2003; Persikov et al 2003). Pro and its derivatives prefer one of

the two major pyrrolidine ring puckers, which are termed C -exo and C -endo.

The ring prefers two distinct twists, rather than envelope like conformations.

C atom experiences large out-of-plane displacements which are designated as

C -exo and C -endo. Pro has a slight preference for the C -endo ring pucker.

A key attribute of Hyp is its imposition of a C -exo pucker on the pyrrolidine

ring via the gauche effect. The C -exo ring pucker pre-organizes the main

e in the YAA position of a triple helix

(Wolff et al 1966; Nishi et al 2005; Shoulders et al 2009, 2010).

1.2.2.5 Side chain interactions

Charged functional groups are generally found in high proportions

in the triple helical domain (15-20%) such as basic functional groups (Lys,

Arg and His) and acidic functional groups (Asp and Glu). Basic functional

groups are observed in YAA position, on the other hand acidic functional

groups are found in XAA position. Interaction between the acidic and basic

side chains are commonly called as ion pairs. It is evident from the literature

that collagen has more tendency to form inter-molecular and intra-molecular

ion pairs than the globular proteins by frequent occurrence of Lys-Gly-

Asp/Glu in the sequence of natural collagen (Schulz et al 1980; Branden et al

1991; Bella et al 1996; Fairman et al 1996; Kwon et al 1996; Chan et al 1997;

Huyghues-Despointes et al 1997; Vekey et al 1997; Yang et al 1997; Kramer

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et al 2000; Periskov et al 2002, 2005a, 2005b; Izumrudov et al 2003;

Maksimiak et al 2003; Perry et al 2003; Heinrichs et al 2005; Sundar Raman

et al 2008; Kirshnamoorthy et al 2011). The formation of ion pairs is

responsible for the enhanced stability of collagen in addition to the canonical

hydrogen bonding interaction. Introduction of ion pairs in short CLPs

increases the stability of the triple helix and helps in self association (Rele et

al 2007). Charged functional groups help in interaction of collagen with

different cell surface proteins. This interaction is very important for physical

strength of cell and for signal transductions regulating the cell differentiation

and proliferation (Sixma et al 1997; Ivaska et al 1999; Emsley et al 2000;

Mizuno et al 2000; Heino et al 2007).

1.3 COLLAGENASE: COLLAGEN DEGRADING ENZYME

Collagenases are specific class of enzymes that hydrolyze

the peptide bonds present in collagen. Based on the source, collagenases are

classified in two categories: (i) matrix metalloproteinases or mammalian

collagenase, (ii) bacterial collagenase.

1.3.1 Matrix Metalloproteinases

Collagenolysis is an integral part of many biological processes,

such as embryogenesis, organogenesis, angiogenesis, skeletal growth, tissue

remodelling, wound healing, arthritis, periodontal disease and cancer

(Robertson et al 1976; Shingleton et al 1996; Greenwald1999; Foronjy et al

2001; Donahue et al 2006). The fibrillar collagens are degraded by a family of

Zn2+ dependent proteases known as matrix metalloproteinases (MMPs).

MMPs recognize specific extra cellular matrix (ECM) component and cleave

them at specific sequence. MMPs cleave many linear substrates but only

MMP-1, 8, and 13 recognize the triple helical structure of collagen and

degrade it. Therefore, they are called as collagenase. Study on the MMP-14

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showed that it also cleaves triple helical structure (Ohuchi et al 1997).

Collagenase cleaves specifically at the 775-776 position (Gly-Ile/Leu bond)

of polypeptide chains, thereby splitting the triple helix into two fragments.

This sequence specificity and substrate selectivity of collagenase

differentiates its function from other MMPs. Structure of type I collagenase is

schematically depicted in Figure 1.4. Active collagenase primarily contains

two domains namely Catalytic (CAT) and Hemopoxin (HPX). The structure

of CAT domain is almost similar to CAT domains of other MMPs. HPX is

also called collagen binding domain. Due to the structural similarity with

hemopoxin, it is named as hemopoxin domain. HPX acts as substrate

recognition domain. This domain has the characteristic four bladed -

propeller structure.

Figure 1.4 1FBL crystal structure of collagenase. CAT domain, HPX

domain and linker regions are shown in red, blue and green colours,

respectively. The metal ions such as Zn2+ and Ca2+ are shown in sky blue

and green, respectively (Courtesy: Jozic et al 2005)

Comparing the size and shape of catalytic sites of collagen specific

proteases and the structure of the collagen, it can be found that catalytic sites

are too narrow to accommodate the triple-helical structure (Fields1991; De

Souza et al 1996; Perona et al 1997; Olsen et al 2001; Fiori et al 2002; Lauer-

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Fields et al 2002, 2009; Jaisson et al 2007; Perumal et al 2008; Ravikumar et

al 2008; Han et al 2010). Therefore, hydrolysis of peptide bonds of collagen

needs to take place through induced fit mechanism by proteases or the region

where collagen does not have triple helical structure (Chung et al 2004). It has

been suggested that the imino acid deficient region i.e. C-terminal to the

cleavage site is important for the binding and cleavage by this enzyme (Fields

1991). This region has a “loose'' helical structure that distinguishes itself from

the rest of the collagen molecule (Fields1991; Stultz 2002). The imino poor

region exhibited the inter-chain distances expected for a standard triple helix,

but it had a greater mobility on slow time scale (Long et al 1993; Liu et al

1996, 1998; Baum et al 1999; Buevich et al 2000, 2001, 2002; Bhate et al

2002; Xu et al 2003). The crystal structure demonstrates that sequence can

influence local conformational changes in triple-helical structure, in terms of

super helical pitch, hydrogen bond, and hydration patterns (Kramer et al

2001). The helical pitch is around 10/3 in imino poor regions and 7/2 in imino

rich regions. The pitch of collagen varies across the domains and types of

natural collagen. It has been hypothesized that the pitch variations might be

an essential factor for collagenase recognition and cleavage (Minond et al

2007).

In the same direction, Stultz et al (2002, 2003, 2006) carried out

various theoretical studies on the CLPs using MD simulation to calculate the

free energy of unfolding for CLPs. Based on the free energy profiles, they

have proposed that imino-poor regions can adopt a low-energy, partially

unfolded state when one of the peptide chains forms a solvent-exposed loop

(Stultz 2006). Similarly, simulations of type I collagen in the imino poor

2 chain is energetically preferred

1 2 chain leads to

the formation of a structure that has disrupted hydrogen bonds at the

collagenase cleavage site. This disruption in hydrogen bonding pattern leads

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2 chain to sample different

2 chain is mediated by

the non-hydroxylation of a proline residue at

(Stultz et al 2003) 1)

2), and points to a critical role for

the non-hydroxylation of proline residues near the collagenase cleavage site.

Recently the same group demonstrated that type I collagen can be degraded at

room temperature; a temperature well below the melting temperature, by

collagenase deletion mutants that only contain the catalytic domain of the

enzyme (Stultz 2002). Based on their findings, collagenase preferentially

recognizes and cleaves partially unfolded states of collagen.

Four major steps are involved in the collagenase activity. (i) initial

binding of HPX domain to triple helix of the collagen, (ii) an unwinding of

the triple helix, (iii) binding of one of the unwound chain of triple helix bind

to the active site of the enzyme and (iv) cleavage of collagen by CAT domain.

The mode of binding of HPX, unwinding of collagen and the binding of CAT

on the unwounded chains are expected to be the key factors in differentiating

the collagenase from other MMPs.

1.3.2 Bacterial Collagenase

Collagenases are used in the nonsurgical treatment of

fibroproliferative disorders such as Dupuytren’s disease, adhesive capsulitis

and Peyronie’s disease. At the same time bacterial collagenases are suitable

for the disaggregation of human tumor, mouse kidney, human adult and fetal

brain, lung and many other tissues, particularly epithelium. It is also effective

in liver and kidney perfusion studies, digestion of pancreas, isolation of non-

parenchymal rat liver cells and hepatocyte preparation. Bacterial collagenase

has found clinical applications in the treatment of third degree burns,

decubitus, and diabetic or arterial ulcers. Bacterial collagenase cleaves -

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chains at sites preceding mostly glycine residues. While many proteases can

hydrolyze denatured collagen polypeptides, clostridiopeptidase A is unique

among proteases in its ability to attack and degrade the triple-helical native

collagen fibrils. Collagenase is specific for the X-Gly bond in this sequence,

Pro/Hyp-X-Gly- where X is the neutral amino acid and they also cleave

denatured collagen and other polypeptides with a related primary structure

(Matsushita et al 2001).

1.3.2.1 Sources of bacterial collagenase

The various sources of bacterial collagenase are Clostridium

histolyticum, Clostridium perfringens, Clostridium capitorale, Pseudomonas

aeruginosa, Mycobacterium tuberculosis, Bacillus cereus, Bacteroides

melaninogenicus, Clostridium tetani, Clostridium welchii, Cytophaga sp.,

Empedobacter collagenolyticum, Geobacillus collagenovergons,

Porphyromona sgingivalis, Pseudoalteromonas mariniglutinosa,

Streptomyces sp., Vibrio alginolyticus, and Vibrio paraheamolyticus (Gibbons

et al 1961; Diener et al 1973; Hanada et al 1973; Appel 1974; Merkel et al

1978; Peterkofsky1982; Hare et al 1983; Chakraborty et al 1986; Tong et al

1986; Endo et al 1987; Makinen et al 1987; Dive et al 1992; Lawson et al

1992; Sasagawa et al 1993; Matsushita et al 1994, 2001; Yu et al 1999; Kang

et al 2005; Itoi et al 2006; Eckhard et al 2009).

1.3.2.2 Bacterial collagenase structure and sequence motifs

Enzymes produced by Clostridium histolyticum have been widely

studied. It is responsible for extensive tissue destruction in gas gangrene, and

its activity is enhanced by calcium ions (Worthington Biochemical Co 1988;

Mookhtiar et al 1992; Willson et al 2003; Matsushita et al 2001). It is known

to produce a mixture of collagenases, which are able to digest the collagen.

The individual collagenases can be divided into two groups. They are grouped

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into class I and class II , and are encoded by distinct

chromosomal genes, ColG and ColH (Yoshihara et al 1994; Matsushita et al

1999) with the molecular masses ranging from 68 to 130 kDa. These ColG

and ColH encode the proteins of 1118 and 1021 AA residues, respectively.

Class II enzymes have cleavage specificity similar to that of mammalian

collagenase. They are able to cleave type I collagen across all three chains

within the macromolecule.

The segmental structures of ColG and ColH are S1, S2, S3a, S3b

and S1, S2a, S2b, S3, respectively, which is shown in Figure 1.5.

Figure 1.5 Segmental structures of two Clostridium histolyticum

collagenases (Courtesy: Matsushita et al 2001)

ColH and ColG are homologous but differ in their segmental

structures (Matsushita et al 1998) and their C-terminal domains are

responsible for collagen binding function (Matsushita et al 2001). ColG

possesses tandem CBDs at its C-terminus, ColH contains only one. These

three domains are homologous to each other, consist of ~110 amino acid

residues and bind to various types of insoluble collagens (Matsushita et al

1998; Toyoshima et al 2001) by recognizing the triple-helical conformation of

collagen (Matsushita et al 2001). The truncation of CBDs from the bacterial

collagenase makes it incapable of hydrolyzing insoluble collagen, but it can

hydrolyze non-triple helical domains and soluble gelatin. C-terminal segments

(S2, S3a, S3b, and S3a3b) of class I collagenase (ColG) are truncated and

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their binding ability on collagen was studied (Matsushita et al 2001;

Philominathan et al 2008, 2009a, 2009b). The molecular masses of S2, S3a,

S3b, and S3a3b are 10.8, 14.2, 13.8, and 27.1 KDa, respectively. A

homologous linker is found at the N-terminus of CBDs, i.e. between S2 and

S3a and between S3a and S3b in ColG, and between S2b and S3 in ColH.

Effect of calcium on binding and collagenolysis can be explored by

using the crystal structure of CBD (S3b) (Matsushita et al 2001; Wu et al

2001; Willson et al 2003; Sides et al 2012). The high-resolution CBD

structure without calcium (apo-CBD) and with calcium (holo-CBD) is shown

in Figure 1.6. CBD forms a -sandwich `jelly-roll' composed of ten -strands

made up of hydrophilic backside and tyrosine-rich face of the protein

structure. It reveals the N- -helix in the

apo -strand in the holo form, which

possibly resulting in domain rearrangement (Wilson et al 2003; Wu et al

2001; sides et al 2012). S3a3b binds to insoluble collagen more efficiently

than S3b, which suggest cooperative binding of the two functional domains to

this macromolecular substrate.

Figure 1.6 Surface representation of the collagen binding interface on

CBD. The binding cleft is colored orange, and Ca2+ ions are indicated

in red spheres (Courtesy: Philominathan et al 2009)

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Based on the crystal structure, Huizinga et al (1997) suggested that

binding of CBD with collagen is achieved primarily through ionic interactions

between negative charged residues on the domain and positively charged

residues in collagen. The most recent solution studies of S3b in complex with

collagenous peptides by small angle X-ray scattering (SAXS), and hetero

nuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR)

titration of various spin-labeled collagenous peptides to 15N-labeled S3b

showed that S3b binds unidirectionally to the C-terminus of collagenous

peptides. The selected CLP are (POG)4POA(POG)5, PROXYL-

(POG)4POA(POG)5, PROXYL-(POG)3POA(POG)6, PROXYL-

(POG)5POA(POG)4, PROXYL-(POG)6POA(POG)3, (POG)4POA(POG)5C-

PROXYL, and PROXYL-(POG)3PCG(POG)4, where PROXYL represents

2,2,5,5-tetramethyl-L-pyrolidinyloxy. The structure of CLP-CBD complexes

is shown in Figure 1.7. CBD binding is not due to non-specific hydrophobic

interactions and it depends on the triple helical conformation (Matsushita et al

2001; Philominathan et al 2008, 2009a, 2009b, 2012). Philominathan et al

(2008, 2009a, 2009b, 2012) indicated the binding of CBD positions to the

under twisted regions of CLP.

CBDs of ColG binds unidirectionally to the C-terminus of the

CLP, which reveals that collagenolysis begins at the C-terminus

(Philominathan et al 2008, 2009a, 2009b; Sides et al 2012). The size of the

binding surface in CBD is 10 Å wide and 25 Å long cleft, the width is

matched with the diameter of triple helix and its length of CLPs.

Tropocollagen binding cleft in solution is made up of Leu924, Ser928,

Arg929, Thr957, Tyr970, Gln972, Leu992, Tyr994, Lys995, and Tyr996.

CBD does not unwind the triple helix, but may help in break up the collagen

fibril.

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Figure 1.7 Structures of CLP:CBD complexes, (A) [PROXYL-

(POG)3POA(POG)6]3:CBD, (B) [PROXYL-(POG)4POA(POG)5]3:CBD,

(C) [PROXYL-(POG)5POA(POG)4]3:CBD, (D) [PROXYL-

(POG)6POA(POG)3]3:CBD, (E) [(POG)4POA(POG)5CPROXYL]3:CBD,

(F) [(POG)4POA(POG)5C-carbamidomethyl]3:CBD. (G) and (H) Two

probable binding modes of [PROXYL-(POG)3PCG(POG)4]3:CBD

(Courtesy: Philominathan et al 2012)

CBD prefers the under-twisted conformation of ((POG)2POA)3 to

that of C-terminal ((POG)3)3, as well as simultaneous binding in C-terminus

also possible. The partial under-twisting positions in CLPs favors hydrogen

bonding interactions from the main chain of amide and carboxyl groups with

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the hydroxyl group of Tyr994 (Wilson et al 2003; Philominathan et al 2012).

Targeting under-twisted regions of tropocollagen may circumvent the energy

barrier required for the unwinding of triple helix. The CBD binding only at

the C-terminal region of the collagen remains still uncertain.

Based on the crystal structure of CBD from ColG collagenase,

interaction pathway of CBD with collagen has been postulated, which

involves three major steps: (i) Anchoring of collagenases onto an insoluble

collagen fibril, (ii) Isolation of single triple helical molecule from bundle, (iii)

Unwinding of triple helix to expose a scissile bond (Matsushita et al 1998,

2001; Holzer et al 2009) or without unwinding the triple helix, collagen-CBD

complex may circumvent the energy barrier required for the unwinding of

triple helix (Philominathan et al 2012).

Most recently Bauer et al (2012) reported the high resolution

crystal structure for S3 CBD from ColH collagenase. The S3 and S3b share

30% sequence similarity. Only three aromatic residues within nine residues in

S3b involved in the substrate binding are conserved in S3. Based on the

sequence alignment, one of the Ca2+chelating aspartates (Asp927) in S3b is

replaced by serine (Ser896) in S3 in the mature collagenase. The structure of

S3 is similar to the structure of S3b from ColG. The S -

sandwich ‘jelly-roll’ -strands. Two calcium

atoms per molecule are found between the linker (861- -strand.

Coordination around the two calcium atoms in S3 are virtually identical with

S3b. S3b and S3 structures implies that the cleft-like shape of the binding

pocket scans the collagen for under-twisted regions and conserved aromatic

residues of the pocket intercalate to the triple-helical collagen without

unwinding its triple helical structure (Philominthan et al 2012; Bauer et al

2012).

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1.3.2.3 Mechanism of collagenolysis

The structure of ColG collagenase was recently resolved by

Eckhard et al (2011) at 2.55 Å resolution. Crystal structure of ColG contains

multiple domains such as an N-terminal domain harboring the catalytic zinc

(Tyr119 to Gly790), a variable number of polycystic kidney disease-like

(PKD-like) domains (Ala791–Asn880), and one or more collagen binding

domains (Glu881–Gly1001, Leu1002–Lys1118), and their domain

organizations are shown in Figure 1.8.

Figure 1.8 Domain organization and architecture of ColG (a) Schematic

of the domain organization of ColG with a functional annotation, (b)

Ribbon representation of the collagenase module S1, with identical color

code as in (a), (c) Full-length model of ColG in complex with a collagen

microfibril (Courtesy: Eckhard et al 2011)

N-terminal collagenase module (catalytic domain-S1) forms saddle

shaped architecture with two domains. The N-terminal saddle flap serves as

an activator domain (Tyr119–Asp388), and comprises an array of 12 parallel

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-helices with tandemly repeated HEAT motifs. A solvent-exposed glycine-

rich linker is positioned at the twist of the saddle seat (Gly389–Tyr397). The

subsequent catalytic sub-domain (Asp398–Gln669) adopts a thermolysin-like

helper sub- -helix pairs of the activator domain and the

catalytic sub-domain combine to form a distorted four-helix bundle and thus

comprise the seat of the saddle topology, which is completed by the glycine-

rich linker. These structural elements latch the relative spatial arrangement of

the peptidase domain and the N-terminal activator, which will turn out to be

critical for collagen binding and triple-helix unraveling. In ColG, catalytic

zinc binding is atypically weak and it is tetrahedrally coordinated by the side

chains of His523, His527and Glu555 as well as a water molecule which was

further hydrogen-bonded to the general base Glu524. This partial zinc

occupancy opens a route for tuning the enzyme activity.

Based on these data, Eckhard et al (2011) suggested integrated

mechanistic model of collagen recognition and processing by ColG

collagenase (chew and digest mechanism): N-terminal activator domain

cooperates with the peptidase domain in both collagen triple helix and

microfibril recognition and processing. The activator and peptidase domains,

forming the two “saddle flaps”, have a distance of ~40 Å, whereas the

diameter of the collagen triple helix is only 15 Å (Figure 1.9). Collagenase

can adopt two preferred conformational states: (i) open state, (ii) closed state.

Closed state allows the collagen triple helix to contact both the activator and

peptidase domain. The closed state is latched by two major contacts at the

bottom of the saddle and by an alternative 4-helix-bundle arrangement at the

saddle seat.

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Figure 1.9 Processing model of triple-helical and microfibrillar collagen,

(a) open conformation: Docked triple helix (green) with peptidase

domain of collagenase, (b) closed conformation: activator HEAT repeats

interacting with the triple helix, (c) semi-opened conformation: exchange

-chains, (d) open conformation:

docked collagenase with microfibril, (e) closed conformation with all

triple helices being expelled from the collagenase, (f) semi-opened

conformation allowing for completely processing the triple helix

(Courtesy: Eckhard et al 2011)

Further the activator HEAT-repeats interacts with triple-helical

collagen, and initiates the unwinding of the triple- -chains which are

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cleaved in processive manner. It reveals that the activator and peptidase

domains remain closed during collagen cleavage, but relax to the open ground

state, once the collagen is degraded.

Collagenolysis is an entropically driven process in a processive

manner. Triple-helical collagen is hydrated by at least two ordered water

layers that are released upon its cleavage, which accompanies the entropy

release. The collagenolysis includes two dimensions: (i) the cleavage of all

collagen chains at one site of the collagen substrate, (ii) multiple cleavage

events along the substrate and it is known as inchworming. The collagen

processing by ColG collagenase occurs from the C-terminus of the collagen

substrate to its N-terminus, consistent with the antiparallel substrate

recognition by the edge strand.

1.4 INTERACTION OF SMALL MOLECULES WITH

COLLAGEN

Protein- ligand interactions are necessary to understand the various

chemical and biochemical processes involved in the living systems.

Crosslinking of various small molecules (crosslinkers) with collagen provides

stability to the collagen towards thermal and proteolytic degradation. In

general, all the crosslinkers interacts with collagen functional groups like –

COOH, -NH2, -OH, -CONH- through covalent amide and imine linkages, H-

bond formation, co-ordinate bond formation, hydrophobic interactions etc.

Crosslinked collagen matrices have intense applications in biological as well

as in industrial field (Stenzel et al 1974; Ramshaw et al 1996). Crosslinked

collagen is preferred as primary source in biomedical applications, mostly

used as biomaterials in blood vessel implants, heart valves, wound or burn

cover dressing and as implant in cosmetic surgery (Stenzel et al 1974;

Ramshaw et al 1996). Stabilized collagen materials are utilized in cosmetic

industries to prevent ageing. In leather industry, stabilization of collagen

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(tanning) is an inevitable process. Many number of crosslinkers have been

developed so far using metal ions (Sreeram et al 2000a, 2000b, 2004; Fathima

et al 2003a, 2003b, 2004, 2005, 2006a, 2006b, 2006c, 2007, 2009, 2010; Usha

et al 2000; Gayathiri et al 2001), polyphenols (Tang et al 2003; Madhan et al

2001, 2003a, 2003b, 2003c, 2005, 2007; Kanth et al 2008; Krishnamoorthy et

al 2008), enzymes (Hand et al 2000; Jones et al 2007; Kang et al 2011),

aldehydes (Madhan et al 2003c; Fathima et al 2004, 2007), oils (Covington

2009), carbohydrates (Kanth et al 2006), iridoids (Covington 2009), epoxides

(Covington 2009), imides (Hafemann et al 2001; Gratzer et al 2007; Usha et

al 2012), cyanates, and sulfochlorides (Covington 2009).

1.4.1 Collagen Preservation: Tanning Agents

Tanning is described as follows:

Conversion of putrescible hide/skin into a non-putrescible

leather against microbiological attack

Change in the long range ordering of collagen, the leather

making protein, resulting in elevation of hydrothermal

stability

Change in the physical appearance and properties, opacity

and handle

Conventionally, stabilization of collagen (tanning) is done by

inorganic and organic tannages by introducing additional crosslinks. Types of

crosslinks formed with collagen are characteristic property of the tanning

materials. Tanning mechanisms have been proposed irrespective of

individual tanning agents based on two aspects. One theory has been proposed

by Covington et al (1997, 1998, 2009) based on thermodynamic parameters

22

such as enthalpic and entropic contributions to the tanning reaction for the

collagen stabilization in the view of hydrothermal stability. Rules can be set

out as follows for the enthalpic contribution to the tanning reaction.

The reaction energy must be maximized and the interaction

must be non-labile interaction between the collagen and

reactant. The bonding should be preferably at least partially

covalent or multiple hydrogen bonding.

The crosslinking must be rigid and short, to impose the

highest degree of structure

Multiple hydrogen bonding involves in structure making

effect which has been achieved by creating a rigid matrix

through in situ crosslinking.

Another aspect has been proposed by Ramasami (2001) based on

the orientation/reorientation of supra-molecular water in the collagen matrix

structure upon tanning reaction leading to hydrothermal stability. The rule is

as follows:

The matrix can be constructed in the presence of reactants as

a covalently linked meshwork, which may disrupt or even

displace the supra-molecular water, because of its higher

energy reaction with collagen.

The matrix must incorporate the supra-molecular water as

the additional level of crosslinking in the case of no

extensive meshwork formation by the tannins interaction

with the protein matrix.

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The creation of a stable, rigid matrix around the collagen,

preferably by displacing the supra-molecular water. This

may incorporate some of the supra-molecular water as part

of the matrix structure.

1.5 MECHANISM OF COLLAGEN DEGRADATION BY

BACTERIAL COLLAGENASE: AN UNANSWERED

QUESTION

Several independent lines have evolved to solve the highly

complex task of collagen binding and hydrolysis. The structure-function

relationship of bacterial collagenases cannot be easily identified due to lack of

crystallographic analysis, which partly arises from the complexity of these

multiple domain architecture. Recently, Eckhard et al (2011) have solved the

crystal structure for ColG collagenase but still information on ColH

collagenase remains elusive. They proposed chew and digest mechanism

based on the enzyme properties using docking studies. CBD targets the under-

twisted regions of tropocollagen in C-terminal region which may circumvent

the energy barrier required for the unwinding of triple helix. But still,

questions regarding CBD of collagenase binding only at the C-terminal region

of the collagen remain. Therefore, much of the mechanistic basis for the

collagenolysis and binding has remained elusive and the questions that need

to be addressed are-

What are the binding forces involved in collagen-

collagenase interaction?

What is the role of chirality (amino acid) in collagenolysis at

the cleavage site of collagenase in collagen?

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1.6 PARAMETERS INVOLVED IN STABILIZATION OF

COLLAGEN AGAINST COLLAGENASE IN THE

PRESENCE OF SMALL MOLECULES: AN UNANSWERED

QUESTION

Leather making mainly concerns the stabilization of collagen

against bacterial collagenase. To address the mechanism of inhibition of

collagen-collagenase interactions by small molecules, several questions need

to be addressed.

Whether the small molecules interact with the

binding/cleavage sites in collagen and stabilize the collagen

against collagenase?

Whether the small molecule binds to the collagenase and

acts as inhibitor to them?

Does interaction of small molecule with collagen alter the

collagen-collagenase interaction via changes in the

functional groups?

Does the binding of small molecule to collagen alter the

collagen-collagenase interaction via alteration in collagen

conformation?

1.7 PROPOSED HYPOTHESIS FOR BACTERIAL

COLLAGENASE BINDING AND CLEAVAGE WITH

COLLAGEN

The present work aims to unravel the binding forces involved in

collagen-collagenase interaction and role of chirality at the cleavage site of

collagenase. The main hypothesis of this thesis is that, the binding forces

25

involved in collagen-collagenase interaction can be identified through

masking of collagen functional groups. This approach will alter collagenase

interaction with collagen in two ways: (i) inhibit the binding of collagen with

catalytic site of collagenase, (ii) inhibit the binding of collagen with CBD of

collagenase, which is necessary to reduce the activation energy required for

peptide bond scission. The role of amino acid configuration at the cleavage

the collagen mimics.

1.7.1 Identification of Binding Forces Involved in Collagen-

Collagenase Interaction

To unravel the contributions of binding forces involved in

collagen-collagenase interaction, protection of reactive amino acid side-chain

functionalities has been carried out via 1-Ethyl-3-[3-dimethylaminopropyl]

carbodiimide hydrochloride (EDC) to understand the contribution of

electrostatic interaction. Tannic acid has been used to study the hydrogen

bonding and hydrophobic interactions.

1.7.2 Role of Chirality (Amino Acid) at the Cleavage Site of

Collagenase in Collagen

The effect of chirality on collagen mimics has been studied using

molecular dynamics approach. To know the sequence specificity and its

chirality on cleavage site of collagenase on collagen, collagen like peptide

models have been used.

1.8 SCOPE OF THE PRESENT WORK

Collagen stabilization against thermal and biological agents plays a

vital role in leather making. The current method used for stabilization of

26

collagen is through interactions with several crosslinkers, in which chrome

and vegetable tannins continue to dominate the tanning process. This is

predominantly due to the lack of understanding in collagenase interaction

with collagen and its inhibition by tanning agents. There is a necessity to

unravel the mechanism of inhibition of collagenase. Identification of forces

involved in collagen-collagenase interaction will provide a platform towards

the structure based design in the selection of crosslinkers for collagen

stabilization. Configurational changes in the amino acid at the cleavage site

will provide a pathway for the development of bio-based tanning system. This

will help to overcome the environmental problems related to the present

chemical based tanning.

1.9 OBJECTIVES OF THE PRESENT WORK

The aim of the present work is to understand the collagen-

collagenase interaction on the basis of identifying the possible forces involved

in the interaction. Possible forces such as electrostatic, hydrophobic, hydrogen

bonding and weak intermolecular forces such as dipole-dipole, van der Waals

interactions are involved in the protein-enzyme interactions. To understand

these forces involved, the present investigation analyses

Electrostatic interaction through blocking the charged

functional groups using chemical crosslinking

Hydrophobic and hydrogen bonding forces using chemical

crosslinking

Role of chirality (amino acid) and sequence importance

through configurational changes in collagen