Skeletal and cardiac muscles are regulated by the tropomyosin/troponin complex. Troponin C (TnC), a subunit of troponin, is the Ca²⁺ receptor that controls regulation. The structure of recombinant skeletal TnC was determined by x-ray crystallography in the four calcium bound "on state" to 2.0 Å resolution.  A comparison of two and four calcium bound TnC structures reveals the interactions that must change during the Ca²⁺-dependent transition.  Sequence variations are shown to account for functional differences in the calmodulin superfamily. In addition, we have solved to 2.0 Å resolution the crystal structure of skeletal TnC complexed with a synthetic peptide which comprises TnI residues Asn96-Lys123. This sequence includes the inhibitory region, TnI(Gly104-Arg115), which can also bind to actin.  The structure reveals the interaction of residues Leu111-Asp119 of TnI with TnC and may represent key structural characteristics of the troponin "on state".  A comparison of the structures of peptide bound skeletal TnC and a drug bound cardiac TnC indicates that the peptide and drug bind to different surfaces of TnC. This information may guide the design of new drugs in the treatment of congestive heart failure and other myopathic diseases.
 
 

1 General Introduction

1.1 How do we move?

1.1.1 Muscle structure and contraction

1.2 Muscle Regulation

1.2.1 Myosin linked regulation

1.2.2 Thin filament regulation

TnC, the calcium-sensitive switch

Cardiac TnC, a drug target

TnC-TnI interactions

1.3 Thesis prelude: 4Ca²⁺TnC with and without peptide

2.1 Introduction

2.2 Results and discussion

2.2.1 Overall structure description

2.2.2 Additional features of the structures

The N lobe of TnC

The C lobe of TnC

Central helix

Calcium binding sites

Crystal packing

2.2.3 The conformational switch

The equilibrium between the open and closed conformations

The switch mechanism

Comparison with CAM

Sequence variations and affinity for Ca²⁺

2.2.4 Comparison of the central helix in TnC and CAM

2.2.5 TnC/TnI interaction

2.2.6 Biological implications

2.3 Materials and methods

2.3.1 Crystallization and data collection

3.1 Overall structure description

3.2 Comparison to other CAM superfamily members

3.3 Stabilization of the central helix by peptide interactions

3.3.1 Packing considerations

3.3.2 Ca²⁺ dependent central linker flexibility

3.3.3 Intrahelical stabilization of the central linker

3.3.4 The CAM analogy

3.3.5 The central linker in solution

3.3.6 Implications of TnI interactions with the central linker

3.4 TnC/TnI interaction mechanism

3.5 Implications for the design of drugs targeting TnC

3.6 Materials and Methods

List of Tables

2.1 Interhelical angles

2.2 Sequence alignment in the central helix/inter-lobe linker region

2.3 Data collection statistics

2.4 Refinement Statistics

3.1 Torsional character of peptide backbone angles

3.2 Intrahelical central linker interactions

3.3 Data collection statistics

3.4 Refinement statistics

List of Figures

1.1 Comparison of TnC and CAM

1.2 Comparison of peptide bound structures of TnC and CAM

2.1 Ribbon diagram of the overall fold of four Ca²⁺ bound TnC

2.2 Comparison of the N and C lobes

2.3 Packing interactions involving Ca²⁺

2.4 Structure of Ca²⁺ binding loops I, II, and III

2.5 TnC packs differently in the two crystal forms

2.6 Conformational switch in the EF-hands of the N-lobe of TnC

3.1 Overall structure of TnC/peptide complex

3.2 Peptide bound to the N lobe

3.3 Central linker stabilization in various structures

3.4 The central helix supports layers of lobes in the crystals

3.5 A mechanism of interaction between TnC and TnI

3.6 An overlay showing possible interactions of cTnI with cTnC and bepridil

3.7 Four structures overlayed: Interactions of TnI with TnC

3.8 Electron density map for TnI[Leu111-Asp119]

 

Many thanks go to my fellow lab members, who helped me in science, in writing, in conversation, and so on. Thank you Yu Li, Jerry Brown, Suet Mui. Special thanks go to Anne Houdusse, who brought her exceptional talents of structural analysis to the problems we faced in Chapter 2, and to Roberto Dominguez, who taught me the practical protein crystallographic method and much more.

Also, thanks go to the Brandeis detector group. In particular, thank you Walter Phillips, Marty Stanton and Charlie Ingersoll, who taught me about the hardware and software of crystallographic data collection. I solved my first structure using data from an x-ray camera that we assembled.

I am very grateful to Zenon Grabarek, our collaborator, who not only provided the protein and peptides that were used in these experiments, but also provided many biochemical insights into the problems we were facing. I am also very grateful Carolyn Cohen, my advisor and teacher, who made all of this possible and gave me a chance to become a scientist. Many Thanks!