COMPUTATIONAL STUDY ON THE STRUCTURE-FUNCTION RELATIONSHIP IN COPPER BINDING PROTEINS

SYNOPSIS

of the Ph.D. thesis to be submitted to the

UNIVERSITY OF MADRAS

In partial fulfillment for the requirements of the award of the degree of

DOCTOR OF PHILOSOPHY

by

V. RAJAPANDIAN Under the guidance and supervision of

Dr. V. SUBRAMANIAN

CHEMICAL LABORATORY CENTRAL LEATHER RESEARCH INSTITUTE

(Council of Scientific & Industrial Research) CHENNAI – 600 020, INDIA

September 2011

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Computational Study on the Structure-Function

Relationship in Copper Binding Proteins

V. RAJAPANDIAN

Chemical Laboratory CSIR-Central Leather Research Institute

Adyar, Chennai 600 020, India

1.0 INTRODUCTION

In bioinorganic chemistry, our focus is on the small part of large

metalloproteins that include the metal ion and its local coordination environment.

Metal containing proteins account for nearly half of all the proteins in nature [1-6].

There are currently over 66, 000 structures in the Protein Data Bank (PDB), and

searching for the keyword “metal” results in over 25, 000 hits. Results obtained from

the search are shown in Table 1 [7]. Metal ions, namely zinc, iron, manganese, and

copper play a vital role in structure, stability, and function of proteins [1-7].

Previous reports showed that in 40% of enzyme-catalyzed reactions, metal ions are

functionally involved [8]. Thus, understanding the metal site in metalloproteins,

metalloenzymes, and metallochaperones has received intensive attention by many

researchers [1-42]. The investigative goal of previous studies was to derive the

relationship between the geometrical structure and its function [1-42].

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Table 1: Metals in the PDB

metal hits metal hits metal hits

Na

Mg

K

Ca

2701

5384

965

5030

V

Cr

Mn

Fe

Co

Ni

Cu

Zn

59

7

1412

1403

337

463

763

5854

Pd

Ag

Cd

Ir

Pt

Au

Hg

8

10

521

2

44

30

343

Several books, book chapters, authoritative review articles and research

articles have appeared on various aspects of metalloproteins, metalloenzymes, and

metallochaperones [1-50]. The usefulness of X-ray diffraction in elucidating the

structures of these systems has been demonstrated in numerous studies [1,2,11-32].

In addition to X-ray diffraction technique, various spectroscopic methods are now

available that provide complimentary insights into the electronic structures of

metalloproteins, metalloenzymes, and metallochaperones [12-15,33-42]. Combined

with electronic structure calculations and molecular dynamics simulations, it is

possible to gain insight into the bonding interactions, the origin of unique

spectroscopic features/electronic structures and catalysis [11-32]. The protein

bound metal site consists of one or more metal ions and side chains of amino acid

residues that define the first coordination sphere of each metal ions [12,13,15]. Such

metal sites can be classified into the following basic types.

(i) structural – configuration (in part) of protein tertiary and/or quaternary

structure

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(ii) storage – uptake, binding and release of metals in soluble form

(iii) electron transfer – uptake, release and storage of electrons

(iv) dioxygen binding – uptake, release and storage of oxygen

(v) catalytic – substrate binding, activation and conversion

The biological significances of vanadium, chromium, manganese, iron,

cobalt, nickel, copper, and zinc are evident from the previous reports [1,2,43-50]. In

fact, the concentration of these transition metals and zinc in human blood plasma is

higher than in sea water [46]. This clearly indicates the biological role of these metal

ions. Iron is the most common transition metal in biology [1,2]. The processes and

reactions in which iron participates are crucial to the survival of terrestrial

organisms, and include ribonucleotide reduction (DNA synthesis), energy

production (respiration), energy conversion (photosynthesis), nitrogen reduction,

oxygen transport, and oxygenation [1,2,43-50]. Other metal ions also participate in

reactions equivalent to those involving iron, however, the activity is less than that of

the native protein with iron [46]. Among various metal ions, copper and iron

participates in many of the same biological reactions [46]. They are:

(i) reversible binding of dioxygen: hemocyanin (Cu), hemerythrin (Fe) and

hemoglobin (Fe)

(ii) activation of dioxygen: dopamine hydroxylase (Cu), tyrosinases (Cu) and

catechol dioxygenases (Fe)

(iii) electron transfer: blue copper proteins (Cu), ferredoxins and c-type

cytochrome (Fe)

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(iv) dismutation of superoxide by Cu and Fe as the redox-active metal (super-

oxide dismutases)

After iron, Cu is the next significantly important element in biological

systems to participate in electron-transfer chains [1,2,50]. Copper is essential for

the survival of living organisms [1,2,50]. It is found in the active sites of copper

binding proteins that copper participates in cellular respiration, antioxidant

defense, neurotransmitter biosynthesis, connective-tissue biosynthesis, and

pigment formation [1,2,43-50]. The thermodynamic and kinetic feasibility of Cu to

oxidize/reduce allows copper-containing proteins to play important role as electron

carriers and redox catalysts in living systems [1,2,43-50]. Since non-bonded Cu(I)

ions are toxic due to their ability to form harmful hydroxyl (OH•) radical, the

intracellular concentration of copper is highly regulated [46]. Both prokaryotic and

eukaryotic cells regulate Cu-homeostasis via dedicated proteins that facilitate its

uptake, efflux, as well as by distribution to target proteins/enzymes [30-50].

Numerous studies have been made to understand the structure and

activity of various metalloproteins, metalloenzymes, and metallochaperones [1-50].

Due to the complexity involved in handling metalloproteins, several model inorganic

complexes have been designed to understand the origin of structure-function

relationship [51]. The development of strategies for computational modelling of

bioinorganic systems requires structural and other reactivity information. In this

context, a huge volume of literature is available on the iron and copper binding

proteins and also on their model systems (inorganic complexes) due to their

amenability for spectroscopic and other experimental investigations [1-51]. In the

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present study, copper binding proteins have been selected to develop a structure-

activity relationship using computational chemistry methods.

Copper Binding Proteins

Copper can occur naturally in its monovalent/reduced form (formal

charge +1) as well as in divalent/oxidized form (formal charge +2) [1,2]. Thus, it is

extensively utilized in redox processes in biological systems [43-50]. It can be toxic

at high concentration in cytoplasm. Cells utilize highly conserved pathways to

manage the uptake, storage, and export of Cu [33-42]. These metal-homeostasis

pathways require high protein−protein specificity, to avoid the mishandling of the

metal and irreversible macromolecular oxidation, and generally involve a Cu

chaperone that binds and delivers Cu to cellular targets [33-50]. In the case of Cu

transport to the secretory pathway, this specificity appears to arise from the fact

that both metallochaperone and target domains share the same fold and metal-

binding motif [33-50].

The pathway essentially involves the transport of Cu(I) to multicopper–

ferroxidase enzyme present in Golgi organelle, mitochondria and thylakoid

organelle [35-39]. In yeast, copper from outside cellular environment is transported

into the cell by two membrane proteins namely Ctr1 (copper transporter) and a

metalloreductase enzyme [37]. After the entry into the cell, Cu(I) is transported to

the Golgi organelles by a metallochaperones Atx1 (anti-oxidant), which hand over

copper to Ccc2 present on post Golgi membrane [35-39]. Ccc2 transports Cu(I)

inside Golgi to the target enzyme multicopper–ferroxidase [37]. There is no clear

mechanistic pathway proposed which is consistent with the observed experimental

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facts. The mechanism is inevitable to understand the designing of copper donor and

acceptor sites by nature to allow the transfer of copper from high affinity domain to

low affinity domain.

Among various copper binding proteins, the blue copper protein is a

special class, which has three properties that differ from what are found in the small

inorganic copper complexes: a strong absorption around 600 nm, a small narrow

hyperfine coupling constant in the electron spin resonance spectrum, and a high

reduction potential [1,2,43-50]. Basically copper proteins have been classified based

on their structure-function and spectroscopic features, which led to the

distinguishing of the Type I, Type II, Type III, Type IV, CuA, CuB, and CuZ systems

[52]. This classification differentiates seven types of active site in the copper

proteins. In the following section, significance of design and engineering of

metalloproteins and metalloenzymes are highlighted.

Design and Engineering

Protein design and engineering have contributed greatly to the

understanding of protein structure and function [9,10,53-55]. A common practice in

this field is to use site directed mutagenesis to replace a specific amino acid in

proteins with other natural amino acids [54]. It has become increasingly evident

that site directed mutagenesis cannot address the precise role of a specific amino

acid owing to the limitation of 20 natural amino acids [54]. It is often difficult to

change one factor, such as an electronic effect, without simultaneously changing

other factors, for example steric effects [53-55]. This limitation is even more evident

in metalloprotein design and engineering, as even a smaller number of natural

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amino acids are capable of coordination with metal ions [9,10,53-55]. More

importantly, the metal-binding sites are intricately designed to accept a specific

metal ion among a large number of other metal ions for binding and activity [53-55].

Because geometry and ligand donor sets of metal-binding sites are much more

varied than those of metal-free active sites, a subtle change of one factor can have a

profound influence on the structure and activity.

An emerging direction of metalloprotein design and engineering is on the

horizon, where unnatural amino acids or non-native metal-containing cofactors are

employed in the design and engineering process [54]. These endeavors have been

shown to be quite effective in elucidating the precise role of key residues in protein

structures and functions, in providing guiding principles on protein design, in fine-

tuning the protein properties to an unprecedented level, and in expanding the

repertoire of protein functionalities [9,10,53-55]. The importance of protein design

and engineering are

(i) to understand the structure and functional relationship

(ii) to design new metalloproteins with novel functions

To carry out design and engineering of metalloproteins and metalloenzymes,

various experimental techniques and computational chemistry approaches have

been used [1,2,9-15,26,51,53-55]. Some of the computational chemistry methods

used in the modelling of proteins in general and metallo-systems in particular are

presented in the following section.

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Methods Used for the Modelling of Copper Binding Proteins

From the computational chemistry perspective, the following methods

are widely applicable in studying metalloproteins, metalloenzymes, and

metallochaperones [11-32,56-59]. These methods are based on both classical and

quantum mechanics.

1) Molecular Mechanics (MM) force field (FF) approach: Development of

new FF parameters and validation

2) Quantum Mechanics(QM)

3) QM/MM

Scheme 1. Various theoretical methods used for the modelling of biological systems.

Both classical and QM based computational methods have been used in

this investigation to predict the conformational changes, dynamics, structure of the

metal binding site, spectral properties, and reaction mechanism of larger systems.

The applications of larger system with QM methods are limited due to the

computational cost. Hence the classical MM based FF methods will be used to

understand the conformational changes and dynamics of the entire metalloproteins

and metalloenzymes.

Limitations of FF methods

The limitations in the generation of new FF parameters for various metal

systems arise from: (i) high symmetry around metal ion and associated multiple

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referencing problem, (ii) charge on metal ion, (iii) different oxidation and spin states

and (iv) geometry optimization of models of the metal site in the metalloproteins

and calculation of vibrational frequencies [27,28]. Therefore the development of FF

parameters is an important step in the modelling of native and engineered

metalloproteins. These parameters have been developed by performing quantum

chemistry calculations.

Limitations of ONIOM methods

Since QM is too expensive for the whole protein, the combined classical

mechanics (MM) and QM approach could be another alternative strategy [60-66]. A

hybrid quantum mechanical/molecular mechanical (QM/MM) method such as

ONIOM (our own N-layered integrated molecular orbital + molecular mechanics)

requires FF parameters for all intra- and intermolecular interactions [60-66]. The

standard quantum chemistry packages contain FF parameters for the protein part

only and thus there is a need for the development of new parameters for the active

site to perform ONIOM calculations. Thus, the development of FF parameters for

copper binding proteins and its validation is one of the main objectives of the

present investigation.

2.0 SCOPE OF THE PRESENT INVESTIGATION

In this study, a systematic attempt has been made to develop FF

parameters for the metal site of copper binding proteins using density functional

theory (DFT)-based Becke’s three-parameter hybrid exchange and Lee–Yang–Parr

correlation (B3LYP) method [67-69]. A set of MM parameters for bond stretching

10

and angle bending and atomic charges for the active site which are consistent with

the AMBER [70] FF have been derived. The new copper amber force field (CAFF)

parameters have been validated by performing MD simulation of copper binding

proteins using AMBER FF for the remaining part of the protein. The structure of the

active site of copper binding proteins has been predicted using ONIOM method

employing newly and existing FF parameters. Both ligand field (LF) and ligand to

metal charge transfer (LMCT) transitions have been calculated using time-

dependent density functional theory (TDDFT) [71] method combined with ONIOM

approach [72]. As mentioned in the previous section, there is no clear

understanding of mechanistic pathway for transfer of Cu from high affinity domain

to low affinity domain. Unraveling such pathway would enhance the understanding

of functions of copper binding proteins and also design and engineering copper

donor and acceptor sites. Thus electronic structure methods (QM) were used to

investigate the copper transfer pathway. All QM calculations were performed by

using the Gaussian 03 suite of programs. MD simulations were performed by using

the AMBER package [70]. The detailed objectives of the present study are given

below

Validation of Existing Force Field

With a view to assess the performance of Extensible and Systematic Force Field

(ESFF) [73] parameters, the following points have been addressed by considering

the structure of model compounds of blue copper proteins and native azurin (AZ)

has been taken as model systems.

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To determine the effect of substitution of different metal ions in AZ

and to compare the geometrical parameters with the corresponding

X-ray structures

To study the consequences of mutation of axial ligand by natural and

non-protein amino acids

Development and Validation of New FF parameters

A systematic attempt has been made to develop new FF parameters

(CAFF) for metal site of native and metal ion substituted AZs using

density functional theory (DFT)-based B3LYP method

MD simulations have been carried out on native, loop contracted,

metal ion substituted AZs in combination with AMBER FF parameters

for the remaining part of the protein to validate the CAFF parameters.

ONIOM Calculation on Copper Proteins

A systematic attempt has been carried out to investigate the effect of

metal ion substitution (Co(II), Ni(II), and Zn(II) instead of Cu(II) ) on

the structure and spectroscopic properties of AZ with complete

protein environment using ONIOM approach

To assess the predictive power of ONIOM approach in treating

metalloproteins as well as to design and engineering of

metalloproteins

Copper-trafficking Pathway

To understand the nature of copper transfer pathway from Atx1 to

Ccc2 system

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To understand the necessary interaction responsible for the

irreversible transfer of copper

To probe the impact of protein environment on the reaction pathway

3.0 STRUCTURE OF THE THESIS

In this section, an overview of the thesis is given, with a short description

of the contents of each chapter.

CHAPTER I

This chapter provides a brief introduction of the structure and function of various

metalloproteins and metalloenzymes along with the necessary details on the

computational methods used in this thesis. The scope of the present investigation is

also provided in this chapter.

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CHAPTER II

In this chapter, a systematic attempt has been made to assess the predictive power

of the ESFF parameters in eliciting the geometries of metalloproteins. The results

show that MM and MD calculations with ESFF parameters can reasonably predict

the structure and overall conformation of native and engineered AZs. Further,

calculated change in strain energies of mutants bear a linear relationship with the

redox potential. This study reveals that ESFF parameters can be used to probe

structure and properties of metalloproteins. Some of the most important structural

features derived from the calculations are given in Figure 1.

Figure 1. Superimposed structures of the crystal and minimized structures of AZ.

(blue, pink and yellow color for crystal, minimized structure of Cu(II) and Cu(I)

respectively)

C112 M121

H117

G45

H46 Cu(II/I)

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CHAPTER III

The third chapter describes the methods which are used to obtain FF parameters.

The force constants of bond stretching and angle bending were derived with the

method proposed by Seminario et al [7]. A brief description of this method is given

in this chapter.

Scheme 2. Schematic diagram for FF derivation.

CHAPTER IV

In this chapter, FF parameters for Type-I, Type-II, Type-III, and binuclear CuA active

sites were derived using Seminario’s method [7]. These parameters were validated

by performing MD simulations on the various copper proteins. The results obtained

from this study illustrate that CAFF parameters are useful to predict the structures

of copper proteins. This study provides a comprehensive set of FF parameters

applicable to a variety of copper-containing biological systems.

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Scheme 3. Schematic illustration for FF derivation and validation.

CHAPTER V

An attempt has been made to investigate the effect of metal ion substitution on the

structure and spectra of AZ using two-layer ONIOM approach. It is evident from the

results that the overall structure of the protein is not altered by metal ion

substitution; nevertheless, the metal ion binding site undergoes noticeable changes.

This chapter highlights the importance of protein milieu in the prediction of

structure, electronic, and spectral properties of native and substituted AZs. This

illustrates the usefulness of ONIOM approach in the designing and engineering of

metalloproteins.

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Figure 2. The ONIOM model consists of 1927 atoms with 56 active site atoms in the

QM (B3LYP/6-31G*) region and the remaining atoms in the MM (Universal Force

Field) region.

CHAPTER VI

Metallochaperone delivers copper ions to specific physiological partners by direct

protein−protein interactions [1,2]. Atx1 (anti-oxidant) is a cytosolic yeast copper

chaperone that delivers a copper atom to the membrane bound ATPase Ccc2 in the

trans-Golgi network [33-42]. In this chapter, the detailed quantum mechanical

calculations for the copper transfer mechanism between Atx1 and Ccc2 proteins has

been carried out to understand the copper trafficking pathway.

Scheme 4. Copper delivery to the trans-Golgi network in yeast.

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CHAPTER VII

In this chapter, summary and conclusions obtained from computational calculations

on various metalloproteins and metalloenzymes are provided. The thesis also

contains a list of references cited in all the chapters.

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5.0 PUBLICATIONS BASED ON PRESENT RESEARCH WORK

1. V. Rajapandian, S. Sundar Raman, V. Hakkim, R. Parthasarathi, V. Subramanian

Molecular Mechanics and Molecular Dynamics Study on Azurin Using Extensible

and Systematic Force Field (ESFF), J. Mol. Struct. (THEOCHEM) 2009, 907, 1–8.

2. V. Rajapandian, V. Hakkim, V. Subramanian, ONIOM Calculation on Azurin: Effect

of Metal Ion Substitutions, J. Phys. Chem. A 2009, 113, 8615–8625.

3. V. Rajapandian, V. Hakkim, V. Subramanian, Molecular Dynamics Studies on

Native, Loop Contracted, and Substituted Azurins, J. Phys. Chem. B 2010, 114,

8474–8486.

4. V. Rajapandian, V. Subramanian, Calculations on the Structure and Spectral

Properties of Cytochrome c551 Using DFT and ONIOM Methods, J. Phys. Chem. A

2011, 115, 2866–2876.

5. V. Hakkim, V. Rajapandian, V. Subramanian, Density Functional Theory

Investigations of the Catalytic Mechanism of β-Carbonic Anhydrase, Indian J.

Chem. 2011, 50A, 503–510.

6. V. Hakkim, V. Rajapandian, V. Subramanian, Catalytic Mechanism of Cobalt

Carbonic Anhydrase: A Density Functional Theory (DFT) Investigation, J. Phys.

Chem. A (accepted)

7. V. Rajapandian, V. Subramanian, A Systematic Study on the Force Field

Parameters for the Copper Proteins, J. Chem. Theory Comput. (to be submitted)

8. J. Vijaya Sundar, V. Rajapandian, V. Subramanian, Probing the mechanism of the

irreversible transfer of copper from Atx1 to Ccc2: A DFT Study, Inorg. Chem. (to

be submitted)

22

6.0 PRESENTATIONS IN CONFERENCES

1. V. Rajapandian, S. Sundar Raman, V. Hakkim, R. Parthasarathi, V. Subramanian,

T. Ramasami, Molecular Modelling Studies on Reconstitution and Mutation in

Azurin, Modern Trends in Inorganic Chemistry (MTIC-XII), Chemistry Dept., IIT

Madras, Chennai. (06-12-2007 to 08-12-2007)

2. V. Rajapandian, S. Sundar Raman, V. Hakkim, R. Parthasarathi, V. Subramanian,

T. Ramasami, Molecular Modelling Studies on Reconstitution and Mutation in

Azurin, International Symposium on Recent Trends in Macromolecular Structure

and Function, Bio-Physics Dept., Madras University, Chennai. (07-01-2008 to 11-

01-2008)

3. V. Rajapandian, V. Hakkim, V. Subramanian, Studies on the Effect of Metal ion

Substitution on the Structure and Spectra of Azurin Employing ONIOM Approach,

Discussion Meeting on Theoretical Chemistry (TCS 2009), Indian Institute of

Science, Bangalore-12. (19-01-2009 to 22-01-2009) (Won the best poster

presentation)

4. V. Rajapandian, V. Subramanian, Electronic Structure and Spectral Properties of

Cytochrome c551, School and Symposium on Advanced Biological Inorganic

Chemistry (SABIC-2009), Tata Institute of Fundamental Research, Mumbai. (04-

11-2009 to 07-11-2009)

5. V. Rajapandian, V. Subramanian, A Systematic Study on the Force Field

Parameters for the Copper Proteins, Theoretical Chemistry Symposium 2010

(TCS10), IIT Kanpur. (8-12-2010 to 12-12-2010)

6. V. Rajapandian, V. Subramanian, Chennai Chemistry Conference 2011 (CCC-

2011), Organized by IIT Madras, CLRI, University of Madras, and Anna

University, held at Department of Chemistry, Indian Institute of Technology

Madras, Chennai, February, (11-02-2011 to 13-02-2011)