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«INVESTIGATING STRUCTURE-FUNCTION RELATIONSHIPS IN FAMILY 7 CELLULASES BY MOLECULAR SIMULATION By Courtney Barnett Taylor Dissertation Submitted to ...»

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INVESTIGATING STRUCTURE-FUNCTION RELATIONSHIPS IN FAMILY 7

CELLULASES BY MOLECULAR SIMULATION

By

Courtney Barnett Taylor

Dissertation

Submitted to the Faculty of the

Graduate School of Vanderbilt University

in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

in

Chemical Engineering

August, 2012

Nashville, Tennessee

Approved:

Clare McCabe Peter T. Cummings Eugene LeBoeuf Kenneth A. Debelak To Mom, Dad, John, and Mal, unwavering in their support and To Trent, for 13 years of patience ii

ACKNOWLEDGEMENTS

This work has been possible through the financial support of the DOE Office of the Biomass Program for funding. Computational time for this research was provided under NSF Teragrid on the TACC Ranger cluster and the NICS Athena and Kraken clusters, and through the resources at NERSC.

I am grateful for the support and guidance I have received during my research from my advisor, Clare McCabe. I am especially grateful for the leap of faith taken when agreeing to let me work from Baton Rouge for nearly my entire graduate career. I would like to thank the members of my Dissertation Committee for their time contributed to this work. Thanks to Faiz Talib for his hard work as an undergrad. I cannot have an acknowledgements section without addressing the group at NREL, especially Dr. Gregg Beckham, who was in essence my co-advisor and instrumental to this work. I thank the rest of the group, Drs. Christy Payne, Mike Crowley, Yannick Bomble, Deanne Sammonds, Lintao Bu, James Matthews, and Mike Himmel, for their massive amounts of patience, assistance, and willingness to answer every rambling email sent their way. They make pretty good friends, too!

Finally, with limited space, I thank my family and friends for maintaining my sanity. To all those I met at Vanderbilt, you are great people. To my Nashville crew (you know who you are), I feel like Dorothy leaving Oz; you all were the best part of this experience, and thanks for walking the road with me. To my closest friends and family that I wish I could name individually, I cannot thank you all enough for your support.

And finally to Trent, bless you bubs, you deserve sainthood at this point; thank you, I love you.

iii

–  –  –

DEDICATION…………………………………………………………………………….ii ACKNOWLEDGMENTS………………………………………………………………..iii LIST OF TABLES……………………………………………………………………….vi LIST OF FIGURES……………………………………………………………………..viii

1. INTRODUCTION

1.1 Background

1.2 Tricoderma reesei Cellulases

Carbohydrate-Binding Module (CBM)

Connective Linker Peptide

Catalytic Domain

1.3 Role of Glycosylation in Cellulase Action

1.4 Summary and Outline of Thesis

2. BACKGROUND AND THEORY

2.1 General Methodology: Molecular Dynamics

2.2 Thermodynamic Integration and Relative Binding Free Energy

2.3 References

3. COMPUTATIONAL INVESTIGATION OF AMINO ACID MUTATION AND

GLYCOSYLATION EFFECTS ON THE CEL7A CBM

3.1 Introduction

3.2 Computational Methods

TI Simulations of Amino Acid Mutations

TI Simulations of Glycosylated CBMs

MD Simulations to Examine the Impact of Glycosylation on CBM Stability..........64!

Simulation Details

3.3 Results

Relative Binding Free Energy from TI

Interaction and H-bonding Between Residues of Interest and Cellulose..................78!

Protein Backbone Fluctuations With and Without Glycosylation

3.4 Discussion

3.5 Conclusions

3.6 References

4. IMPACTS OF O-GLYCOSYLATION ON THE STRUCTURE AND FUNCTION OF

CBMS IN FUNGI AND YEASTS

iv

4.1 Introduction

4.2 Computational Approach

Thermodynamic Integration

All-atom MD Simulations of Glycosylated CBMs

4.3 Results

Relative Binding Free Energy from TI Simulations

CBM Glycoprotein-Cellulose Interaction Energy

CBM Protein Backbone Root-mean Square Deviation (RMSD) and Root-mean Square Fluctuation (RMSF)

CBM Glycoprotein-Cellulose Hydrogen Bond Analysis

4.4 Discussion

Binding Affinity Differences in Dimer Glycoforms

Binding Affinity Differences in Branched and Linear Trimer Glycoforms............125!

Impact of Glycosylation Location on Protein Structure and Affinity

Glycosylation As a Natural Means to Improve Binding Affinity

4.5 Conclusions

4.6 References

5. BINDING SITE DYNAMICS AND AROMATIC-CARBOHYDRATE

INTERACTIONS IN PROCESSIVE AND NON-PROCESSIVE FAMILY 7

GLYCOSIDE HYDROLASES

5.1 Introduction

5.2 Computational Procedures





All-Atom MD Simulations of the Cel7A and Cel7B Catalytic Domains................144!

Thermodynamic Integration and Relative Cellodextrin Binding Free Energy........149!

5.3 Results

Molecular-level Comparison of the Cel7A and Cel7B Wild Type Catalytic Domains

Molecular-level Comparison of the Bound Cellodextrin in the Cel7A and Cel7B Wild Type

Relative Binding Free Energy from Aromatic Acid Mutation in the Cel7A and Cel7B from TI Simulation

Molecular-Level Comparison of Aromatic Acid Mutations

5.4 Discussion and Conclusions

5.5 References

6. CONCLUSIONS AND FUTURE WORK

6.1 Conclusions

6.2 Future Work

Validation of Findings on Glycosylation Impacts to Binding Affinity

Processive and Non-processive Catalytic Domains in Other GH Families.............189!

6.3 References

v LIST OF TABLES

Table 1.1: Sequence alignment of various CBMs for exoglucanases (Cel7A and Cel6A) and endoglucanases (Cel7B) in example fungi [24].

The flat-face residues of interest are highlighted in yellow. The native, or wild type, Cel7A amino acid sequence is shown on the first line

Table 1.2: Linder et al.

calculated partition coefficients and binding free energy for Cel7B over Cel7A wild type CBM and mutations in the Cel7A wild type CBM planar face [28,29]. Amino acid lettering: Y, tyrosine; W, tryptophan;

N, asparagine; A, alanine

Table 3.1: Relative binding free energy (!!G, kcal/mol) from amino acid TI calculations, including the two containing native glycosylation (Y5A-G and Y5W-G), and associated change in partition coefficient (KMut/KWT-NG).

The partition coefficient change was not calculated for the intermediate steps, Y5A-G and Y5W-G. The S3M1+Y5A-G and S3M1+Y5W-G entries are the sum of the Y5A-G and Y5W-G and S3M1 (see Table 3.4) entries, respectively

Table 3.2: Relative binding free energy (!!G (kcal/mol)) of Y5A, F5A, Y5F calculated (Equation 3.

2) and Y5F actual from 11 electrostatic windows and 13 van der Waals windows

Table 3.3: Relative binding free energy (!!G (kcal/mol)) of Y5W, S3M1, Y5WG calculated (Equation 3.

3) and Y5W-G actual

Table 3.4: Relative binding free energy (!!G, kcal/mol) from the native glycosylation TI calculations, for a single O-mannose residue at Ser-3 (S3M1) and a disaccharide mannan at Ser-3 (S3M2), and associated change in partition coefficient (KMut/KWT-NG).

The S3M1+S3M2 entry is the sum of the previous two entries

Table 3.5: Relative binding free energy (!!G, kcal/mol) from the engineered glycosylation TI calculations, with a single O-mannose residue at Ser-14 (S14M1-NG) with no glycosylation and a single O-mannose residue at Serwith the native glycans present (S14M1), and associated change in partition coefficient (KMut/KWT-NG).

The S3M1+S14M1 entry is the sum of the S14M1 and S3M1 (see Table 3.4) entries.

Table 3.6: Number of unique hydrogen bonds formed between the mannose residue of interest and the cellulose surface during the 100 ns MD simulations using a hydrogen bond cutoff of 3.

0 Å and an angle criteria of 60° from linear. The typical duration of each bond was between 10 and 15% of the total run.

vi Table 4.1: Cumulative relative binding free energies as a result of changing glycan patterns compared to a non-glycosylated wild type CBM.

The entire glycoform is the mutation from the non-glycosylated wild type, with green and blue representing mannose and glucose residues respectively.

Table 4.2: Individual TI simulation results corresponding to structures in Figure

4.2. The electrostatic and VDW values are calculated using Equation S1, i.e.

!!GElec = !GBound - !GFree. Partition coefficient ratios were not calculated for individual simulations

Table 4.3: H-bond duration between the glycans and cellulose surface.

H-bonds are calculated using a distance cutoff of 3.0 Å and an angle criteria of 60° from linear. The duration (% of run) is calculated by counting the number of bonds and dividing by the total number of observations. Refer to Figures 4.7 and 4.8 for graphical representations of the number of bonds.

Table 5.11: Count of residues involved with H-bonding to a cellodextrin predicted by structural studies [8,15] and the current study’s simulation results.

..................155!

Table 5.12: Hydrogen bonding patterns and occupancies in Cel7A and Cel7B wild type CD.

Where applicable, active site residues are listed first in red (GluGlu-196, Asp-214/Asp-198, and Glu-201/Glu-217), followed by key aromatic tunnel residues in blue (Trp-376/Trp-329, Trp-367/Trp-320, TrpTyr-38, and Trp-40/Trp-40) for Cel7A/B. Other than the catalytic and aromatic residues, only occupancies 5% of the total 250 ns run are displayed

Table 5.13: Cel7A wild type native contact residues and fraction by binding site.

VDW and electrostatic interaction energies shown with errors calculated using block averaging

Table 5.14: Cel7A wild type native contact residues and fraction by binding site.

VDW and electrostatic interaction energies shown with errors calculated using block averaging

Table 5.15: Relative binding free energy changes for Cel7A and Cel7B per binding site as a result of aromatic acid mutation.

The partition coefficients were estimated using Eqn 5.1 for experimental binding affinities and then inverted to show the improvement of the wild type over the mutated states..........165!

Table 5.16: Detailed relative binding free energies and associated binding affinity (KWT/KMut) calculated from TI for the T.

reesei Cel7A system

Table 5.17: Detailed relative binding free energies and associated binding affinity (KWT/KMut) calculated from TI for the T.

reesei Cel7B system

–  –  –

Figure 1.1: Energy Consumption in the United States in 2010 by sector (Quadrillion BTUs) [2]

Figure 1.2: Cellobiose units (a) form cellobiose chains (b) containing hydrogen bonding networks, shown with red dotted lines.

The chains and sheets combine to form 36-chain microfibril structures (c).

Figure 1.3: Simplified schematic of synergistic cellulase enzyme action on crystalline and amorphous cellulose as suggested by Lynd et al.

[18] Nonreducing ends (white box) are drawn to the left and reducing ends (black box) are drawn to the right. In Trichoderma reesei, Cel7A is a reducing end exoglucanase, Cel6A is a non-reducing end exoglucanase, and Cel7B is an exoglucanase

Figure 1.4: Rendering of the Cellobiohydrolase-I (Cel7A) enzyme in complex with a cellulose fibril.

A cellodextrin chain (red) is threaded into the CD where hydrolysis will take place. The connective linker is heavily glycosylated with mannose sugars (light blue). Rendering taken from Himmel et al., 2007 [9].

Figure 1.5: Proposed catalytic cycle of Cel7A on crystalline cellulose from Chundawat et al.

, 2011 [3]. The yellow and blue space-filling representations are O-glycosylation on the linker and CBM and N-glycosylation on the CD, respectively. The light blue molecule is the Cel7A enzyme, and the green substrate is a cellulose microfibril. The cellobiose product is expelled in (f) and shown in pink

Figure 1.6: Cel7A Family 1 CBM with flat face residues shown.

Aromatics (TyrTyr-31, and Tyr-32) are shown in yellow and polar (Asn-29 and Gln-7) are shown in orange

Figure 1.7: O-glycosylation cites of Cel7A linker peptide as proposed by Harrison [44] and Nevalainen [19] with first three residues of CBM included.

Mannose residues are represented by green circles. All of the Thr and Ser in Cel7A on the linker peptide are glycosylated with 1 to 3 mannose sugars with evidence of sulfation and phospohorylation also occurring in some strains [19,44,48].

Figure 1.8: Comparison of Cel7A (A and B) and Cel7B (C and D) catalytic structures [25,59].

The entrance to the Cel7A tunnel is shown in panel B and the entrance to the Cel7B cleft is shown in panel D. The protein loop structures that form the tunnel in Cel7A and the shorter loop structures present in the Cel7B cleft are both shown in blue. The cellodextrin is shown in green.

viii Figure 2.1: Graphical representation of the thermodynamic paths used to calculated !!G in experiment (top to bottom) and alchemically via computation (right to left). WT represents the wild type and Mut represents the mutation.

Figure 2.2: Example Lennard Jones (van der Waals) potential curves generated from equation 2.



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