Folding and Design of Helical Repeat Proteins Prof. Lynne Regan
Folding and Design of Helical Repeat Proteins
Prof. Lynne Regan Molecular Biophysics & Biochemistry Dept. Yale Univ ersity New Hav en, CT. U.S.A.
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Tetratricopepti de Repeat (TPR)
HEAT repeat
Ankyrin repeat
Leucine rich repeat 2
Tetratricopepti de HEAT repeat Leucine rich repeat repeat (TPR)
Ankyrin repeat
To better understand the behavior of natural proteins
To create novel proteins with interesting new activities 3
The screen versions of these slides have full details of copyright and acknowledgements 1 Folding and Design of Helical Repeat Proteins Prof. Lynne Regan
Repeat Protein vs. Globular Protein
110 100 100 90 90 80 80 70 70 60 60 50 50 Sequence 40 Sequence 40 30 30 20 20 10 10
10 20 30 40 50 60 70 80 90 100 110 10 20 30 40 50 60 70 80 90 100 Sequence Sequence
Predominance of short range interactions Repetitive, regular structure
A more tractable protein folding problem? 4
Tetratricopeptide Repeat (TPR)
34 amino acid repeat Single TPR shown in green
TPR2A domain of HOP binding its peptide ligand
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Genomic Distributions of Proteins with Different Numbers of Tandem TPR Repeats
Homo Sapiens MODELS Saccharomyces Cerevisiae A.Thaliana Bacteria Average % of TPR of repeats %
Number of TPR repeats
STRUCTURE 6
The screen versions of these slides have full details of copyright and acknowledgements 2 Folding and Design of Helical Repeat Proteins Prof. Lynne Regan
Consensus Design of a TPR Motif Global Global propensity
Position in the TPR motif
High conservati on in core and inter repeat residues Large small packing and automatic ‘co variati on’
Proteins: N cap (CTPR) n capping helix 7
CTPR1, CTPR2 & CTPR3
Crystal structures of CTPR2 & CTPR3
CTPR3 is considerably more stable Stability increases with increasing than natural 3 TPR proteins (TPR2A) number of tandem repeats
[GuHCL] (M) 8
Introducing Co Variation, Enhancing Stability
CTPR3* has estimated Tm of 103 oC in absence of denaturant
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The screen versions of these slides have full details of copyright and acknowledgements 3 Folding and Design of Helical Repeat Proteins Prof. Lynne Regan
Can We Describe the Stability of TPR Proteins by a Simple Model?
Dominance of nearest neighbor interactions Increase in stability with number of repeats Increase in cooperativ ity with number of repeats
[GuHCL] (M)
1D Ising model (Zimm Bragg) Treat each helix as a unit, for which the energy difference between U and F = ‘H’ How well does such All units a simple are identical model describe and thus hav e the same v alue of ‘H’ the measured data for a series of TPR proteins? Cooperativ ity from interactions between neighboring units, a coupling energy, ‘J’ Assume all couplings are identical, and therefore hav e the same v alue of ‘J’ 10
Make More Proteins!
Etc., etc. etc .
CTPR1 to CTPR20 11
Stability as a Function of Number of Tandem TPR Repeats
Works equally well with thermal denaturation data
Applicable to different TPRs with different v alues of ‘J’ & ‘H’
Predicts the behav ior of all TPRs within a series from data on a subset
Symbols: Experimental data Solid lines: Fits to Ising model 12
The screen versions of these slides have full details of copyright and acknowledgements 4 Folding and Design of Helical Repeat Proteins Prof. Lynne Regan
Predictions of the Ising Model
Significant populati on of partially folded species not ‘2 state’ denatur ati on 13
Protein Stability on a Residue Specific Basis
Amide hydrogen exchange N N (ppm) N N (ppm) 15 15 – – 2 2 ω ω
10 9 8 7 10 9 8 7 1 1 ω2 – H (ppm) ω2 – H (ppm)
Measure the rate of exchange of each proton with solvent Compare the measured rates with the intrinsic ‘unfolded’ exchange rate of each proton Calculate a ‘protection factor’ for each proton 14
Protein Stability on a Residue Specific Basis Protection factor
Sequence
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The screen versions of these slides have full details of copyright and acknowledgements 5 Folding and Design of Helical Repeat Proteins Prof. Lynne Regan
Other Series and Different Behaviors Explained in Terms of the Ising Model
0 2 4 6 0 0 1 0 2 4 16
But What Do the Long TPRs Look Like?
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Structure of CTPR8
80 Å
35 Å
One turn equals exactly 8 repeats 18
The screen versions of these slides have full details of copyright and acknowledgements 6 Folding and Design of Helical Repeat Proteins Prof. Lynne Regan
Structure of CTPR8
Towards a molecular basis for H and J
Places limits on possible models or ligand binding by long, natural TPRs
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Super Position of TPR Domain of OGT and CTPR8
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Functional Design: Ligand Binding
TPR1 binds C terminus of Hsp70
TPR2A binds C terminus of Hsp90
Concave ligand binding face
Convex ‘back face’
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The screen versions of these slides have full details of copyright and acknowledgements 7 Folding and Design of Helical Repeat Proteins Prof. Lynne Regan
Functional Design:
Specific contacts
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The Nature of Ligand Binding Sites in Proteins
Conv entional wisdom
Buried residues are most conserv ed, they specif y the f old
Surf ace residues are most v ariable, less important
Conserv ed surf ace residues imply f unctional importance
But should this apply to proteins which use the same framework but which bind a v ariety of different ligands? 23 Global Global propensity
Position in the TPR motif Conserved hydrophobic residues specify the fold But what about the non conser ved residues? How can we better quantify variability?
SEQUENCE ENTROPY calculatio ns at each position
How variable is each position relative to a reference state? 24
The screen versions of these slides have full details of copyright and acknowledgements 8 Folding and Design of Helical Repeat Proteins Prof. Lynne Regan
Hypervariability Defines the Ligand Binding Site
Concave, front Convex, back face ligand binding face
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Analogous to Antibody Hypervariable Regions
Wu and Kabat, 1970 26
Hypervariability Defines the Ligand Binding Site
Concave, front Convex, back face ligand binding face
But is this a general result? 27
The screen versions of these slides have full details of copyright and acknowledgements 9 Folding and Design of Helical Repeat Proteins Prof. Lynne Regan
Yes!
Ankyrin repeats
Zinc fingers
PDZ domains
And others in progress .
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ANK Repeats
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Andreas Pluckthun
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The screen versions of these slides have full details of copyright and acknowledgements 10 Folding and Design of Helical Repeat Proteins Prof. Lynne Regan
Functional Design
TPR2A 1. Specific contacts 2. Indirect electrostatic optimizati on
Negative Target peptide: C terminal of Hsp90 MEEVD COOH Neutralized
Positive
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Long Range Electrostatic Interactions Modulate Binding Affinity
CTPR390+B (Kd=1µµµ M)
CTPR390 (Kd=200 µµµ M)
CTPR390 B
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Binding Is Specific
Hsp90
Hsp70
CTPR390 + B binds Hsp90 more tightlyand with greater discrimination against the non cognate ligand than does the natural TPR2A domain of HOP
Useful for dissecting the mechanism of Hsp90 mediated folding and degradation 33
The screen versions of these slides have full details of copyright and acknowledgements 11 Folding and Design of Helical Repeat Proteins Prof. Lynne Regan
‘The Awesome Power of Genetic Screens & Selections’ Split GFP detection of protein protein interactions
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Split GFP Reports on Affinity & Specificity
TPR2A Hsp90 interaction can be detected by split GFP in mammalian cells
R2 R2 R2 R2
Vector control GFP Split GFP Non cognate TPR2A Hsp90 pairs 35
Identifying TPR Domains with Novel Binding Specificities
MAKQALKEKELGNDAYKKKDFDTALKHYDKAKELDP TPR2A MAKQALKEFELGKDAHKKKDFDTALKHYDKAKELDP MAKQALKEFELGMDAHKKKDFDTALKHYDKAKELDP MAKQALKEIELGRDAHKKKDFDTALKHYDKAKELDP 36
The screen versions of these slides have full details of copyright and acknowledgements 12 Folding and Design of Helical Repeat Proteins Prof. Lynne Regan
Summary Repeat proteins versus globular proteins Consensus design, the role of conserved hydrophobic residues Co variation can modulate stability, but is not essential to specify a stable fold Structure and stability of the designed CTPR proteins Understanding the thermodynamic behavior of repeat proteins in terms of a 1D Ising model Predictions of the Ising model Amide H exchange monitored by NMR to study stability on a residue specific basis The structure of long TPRs Hypervariability defines the ligand binding site: a general result Functional TPR designs: specific contacts to the peptide ligand AND long range electrostatic interactions Useful designer proteins ‘The Awesome power of screens and selections’ combined with design to expand the repertoire of novel proteins 37
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The screen versions of these slides have full details of copyright and acknowledgements 13