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Proteins:Proteins:Evolution & DesignEvolution & Design

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Outline● What have we learned from nature protein evolution→

– Permanence of protein in evolution● Reconstruction of evolutionary history

– Change of protein in evolution

● How can we apply the knowledge protein design→– Computational design– Design of proteins– Design of enzymes

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Similarities suggests evolutionary relationships

Similarityin

SequenceStructure

Function / mechanism

Homology

Analogy

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Sequence determines structure

Sequence specifies structure

C. B. Anfinsen, 1950s

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Protein sequence space is BIG

A2A2A1A1

A3A3

A4A4

A5A5

A6A6 A7

A7

20x 20

x 20

x 20

x 20…

Each of the 20 amino acids can be at each position of the polypeptide chain

→ 20Nres protein sequences

A polypeptide of 100 AA → possibilities > num of particles in the universe

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Most polypeptides don’t fold

Enormous number of possible amino acid sequences

Only a tiny subset fold reliably into a functional

native state

Keefe and Szostak (2001) Nature 410Wei et al (2003) PNAS 100

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Levinthal's paradox● Protein conformational space is huge

– Two torsion angles (Φ,Ψ) per residue– m values per torsion angle– m2Nres possible backbone conformations

● Proteins folds quickly– Within a few ms or s

wikipedia

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How did proteins originate?

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Evolution is cumulative● Cumulative selection

– Evolution is not a completely random search

– Partially correct intermediates are retained

● Eventually, they build up to large, stable folds– Duplication, fusion,

recombination, accretion

Berg JM et al. Biochemistry. 5th ed.

# of

ke

ystro

kes

1010

Protein evolution is cumulative● “Correctness”

– Native proteins are only marginally stable● ca. 0.1 kcal/mol per residue

– Intermediates are not necessarily stable on their own

● Rather, they show signifcant structural preference

– Worked as cofactors to ribozymes● Assume preferred structure upon binding

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SsSs shared between diferent folds

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“Piecing together the evolution of proteins”

Alva et al. (2015) eLife

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Reconstruction of Evolutionary Events

● Evolution cannot be proven– any direct observation of intermediate forms is impossible

● We study the likelihood of certain types of events and use it to extrapolate from traits observed today– We use protein sequences as documents of evolutionary events

● Why can we extrapolate so far back in time?– Because of the high evolutionary permanence of protein

domains● Sequence / structure / function / mechanism

Lupas & Koretke (2008) Evoluton of protein folds

...

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Permanence of proteins across timeUbiquitin

human UBI MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG 7696% id |||||||||||||||||| |||| ||| ||||||||||||||||||||||||||||||||||||||||||||||||yeast UBI MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG 7613% id | | | | | | | | ||yeast SUMO INLKVSD-GSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG 86

N N

C CHuman & yeast UBI● diverged > 2 billion yrs ago● vital for eukaryotic

protein degradation

Yeast UBI & SUMO (paralogs)● only 13% seq identity● but key residues conserved

(found by profle-based methods, e.g. PSI-BLAST)

Lupas & Koretke (2008) Evoluton of protein folds

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Permanence of proteins across timeRibosomal proteins

Lupas & Koretke (2008) Evolution of protein folds

The sequences of core ribosomal proteins are still more than 40% identical between all organisms.

So central to cellular processes that its modifcation has become nearly impossible

→ living fossil

David Goodsell & RCSB

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Permanence of proteins across timePoint mutations do not alter fold

● Hundreds of crystal structures of wild-type proteins and their mutants show that point mutations generally do not alter the fold of a protein.– e.g. look at each SCOP

family N

C’N’

1 mutation

NC

C’N’

RNA-binding protein ROPLefthandeee & andtiparallel

(PDB 1ROP)

Rightthandeee & mixedparallel & andtiparallel bundle

(PDB 1B6Q)

Glycos, Cesareni & Kokkinidis (1999) Structure 7:597

A31P

● Exceptions do exist.

e.g. coiled-coils and helical bundles

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Reconstruction of ancient protein sequences

● It allows to investigate – the evolutionary past

of present-day protein structure and function directly in the lab

– evolutionary pathways– adaptive selection– functional divergence

● Suggested by Pauling and Zuckerkandl in 1963

● Only become feasible recently due to development of – genome sequence databases – phylogenetic inference methods

● Methods: – Parsimony– Maximum likelihood– Bayesian inference

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Horizontal vs. vertical analysis of protein families

Harms & Thornton (2010) Curr Opin Struc Biol,20:360

The functional change was caused by a subset of sequence changes along branch C (black box), while permissive mutations on branch B (star) were required to allow the protein to tolerate the function-switching mutations. Restrictive mutations incompatible with the ancestral function accumulated on branch D (cross). Swapping residues between modern proteins (arrow) is inefficient because the sequences differ by all mutations along A, B, C, and D. Protein X does not have the permissive mutations and cannot take on the derived function, while protein Y has restrictive mutations that do not allow it to tolerate the ancestral function.

ancestral function (flled circle) derived function (open circle)

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Origin and evolution of thermophilyAncestral Reconstruction of LeuB

Thermophily is thought to be a primitive trait, characteristic of early forms of life on Earth

Optimal growth temperatures: 20 °C25–30 °C 37 °C45–50 °C60–80 °C

Hobbs et al. (2012) Mol Biol Evol,29:825

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Origin and evolution of thermophily Ancestral Reconstruction of LeuB

Hobbs et al. (2012) Mol Biol Evol,29:825

Trend in thermal adaptation for reconstructed ancestral LeuB enzymes over evolutionary time/estimated age.

Unusual catalytic properties of ANC4 compared with other ancestral LeuB enzymes.

‘Structural analysis suggests that the determinants of thermophily in LeuB from the LCA of Bacillus and the most recent ancestor (ANC1) are distinct and that thermophily has arisen in this genus at least twice via independent evolutionary paths.’

ANC4

ANC1ANC3

ANC2

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Fold change in evolution● Fold space is small

– Thus, structure similarity more likely implies analogy than sequence similarity

● Homologous proteins could also show major structural difference

● Mechanisms– Insertions / Deletions– Circular permutations– Point mutations– Topological substitutions

Swaps & strand invasions– Duplication & fusion– Environment

Grishin (2001) JSB,134:167

Substitutions of SSEs

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Mechanisms of fold changeDeletion

N

CN

C

deletion

Bacterial luciferase(PDB 1LUC)

Nonfuorescent favoprotein(PDB 1NFP)

Grishin (2001) JSB,134:167

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Mechanisms of fold changeCircular permutation

N

C

N

C

circular permutation

C2 domain of synaptogamin I(PDB 1RSY)

C2 domain of phospholipase C(PDB 1QAS)

Grishin (2001) JSB,134:167

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Mechanisms of fold changeCircular permutation

Tandem, in-frame gene duplication:

Loss of stop codon:

3' deletion:

Resolution through further deletionI: Gene returns to previous state

II: Circular permutation

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Mechanisms of fold changeTarget binding

Transcription factor NusGNTD & CTD independent

(PDBs 2K06 & 2JVV)

Transcripton factor RfaH (paralog of NusG)CTD shields RNApol-binding site in NTD

(PDB 2OUG)

N

C

N

C

Belogurov et al. (2007) Mol Cell,26:117

Dissociate upon DNA binding

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Mechanisms of fold changeTarget binding (cont)

Transcripton factor NusGNTD & CTD independent

(PDBs 2K06 & 2JVV)

N

C

N

C

Burmann et al. (2012) Cell

X-ray structure of RfaH(PDB 2OUG)

NMR structure of RfaH-CTD(PDB 2LCL)C

Switch friom a transcription tio a translation factior: Upon

interacton with its target DNA the CTD opens up a RNAP-binding site

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Mechanisms of fold changeDuplication, diferentiation & swapping

duplication and diferentation

3D diomain swapping

decioration, duplication and diferentation

C’N

N’

C

N

AAA+ C-domain(PDB 1IN4)

ClpA N-domain(PDB 1K6K)

Histone dimer(PDB 1B67)

CN C

Alva et al. (2007) BMC Struc Biol,7:17

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Fold change based on environmentLymphotactin

N

C(66)

N

C(60)

C’(60)N’

salt/temperature

monomeric chemokine fold(PDB 1J8I)

Agonist of G-protein coupled XCR1 receptor

dimeric beta-sandwich fold(PDB 2JP1)

Binds glycosaminoglycans

Under normal conditions lymphotactin exists in both forms. The equilibrium between these two states can be changed by varying salt and temperature conditions. Other chemokines are restricted to a single conformation by two conserved disulphide bonds, one of which is absent in lymphotactin. Upon engineering of this disulphide bond into lymphotactin, it is locked in the monomeric state

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Fold change based on environmentInfuenza hemagglutinin

N

C

N

C

pH

a-helical homotrimer(PDB 1HGG)

Three-helix bundle(PDB 1HTM)

Mediates membrane fusion

Bullough et al. (1994) Nature,371:37

Under acidic conditons, hemagglutnin rearranges by extending the N-terminal a-helices of the monomers. The rearranged helical segments form a long three-helix bundle. The change mediates fusion of the viral and host cell membranes.

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Fold change through engineering

Alexander et al. (2009) PNAS,106:21149

GA95albumin-binding 3a fold

(PDB 2KDL)

GB95IgG-binding 4b+a fold

(PDB 2KDM)

N

C N

C

mutation

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Fold change through engineering

Alexander et al. (2009) PNAS,106:21149

albumin-bindingGA77

IgG-bindingGB77

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Outline● What we have learned from nature protein evolution→

– Permanence of protein in evolution● Reconstruction of evolutionary history

– Change of protein in evolution

● How can we applying the knowledge protein design →– Computational design– Fold change by engineering– Enzyme design

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Protein designThe inverse folding problem

Sequence determines structure

Design sequence fora predefined structure

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Native structures are conformationalfree energy minima

Ener

gy

native

unfolded

Protein design:With which seq is this an energy minimum?

Anfinsen's dogma:

The native structure corresponds to a

1.unique2.stable3.kinetically accessible

free energy minimum.

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Conformational degrees of freedom

Figure: Harder et al. (2010) BMC Bioinformatcs

Degrees of freedom in glutamic acid:• Backbone dihedrals Φ and Ψ,

• Side chain dihedrals Χ1, Χ2, Χ3,

→ ~32xNres polypeptide conformations

If Nres = 100● 3198 possibilities

→ folding time > the age of universe

● Obviously paradoxical to folding time● ms or μs

→ Levinthal’s paradox

Ca

CbCg

Cd

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… and many possible amino acid combinations

A2A2A1A1

A3A3

A4A4

A5A5

A6A6 A7

A7

20x 20

x 20

x 20

x 20…

Each of the 20 amino acids can be at each position of the polypeptide chain

→ 20Nres protein sequences

A polypeptide of 100 AA → possibilities > num of particles in the universe

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Abstractions in energy calculationsMolecular mechanics

● Nuclei and electrons are lumped into atom-like particles● Atom-like particles are spherical (radii obtained from

measurements or theory) and have a net charge (obtained from theory)

● Interactions are based on springs and classical potentials ● Interactions must be pre-assigned to specifc sets of atoms● Interactions are transferable between different molecules● Interactions determine the spatial distribution of atom-like

particles and their energies

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Molecular mechanicsForce feld

Atoms spheres→Bonds springs (can stretch, bend, or twist)→

Non-bonded interactions:● van der Waals attractions● electrostatic attractions/repulsions

Energy = Stretching Energy + Bending Energy + Tiorsiion Energy + Nion-Bionded Interaction Energy

● Absolute quantities have no meaning● The differences between

conformations have meaning

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Quick energy calculationRosetta design

Figure: D.Baker, youtube

(1) Lennard-Jones Potential, prefers atoms close, but not too close

(2) Implicit solvation model (penalizes polar residues in the core of a protein)

(3) Hydrogen bonding (allows polar residues in the core of the protein)

(4) Close electrostatic interactions (5) Preference of torsion angles

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Rotamers & conformers● Conformational isomers can be inter-converted by

rotations around single bonds

● Rotamers are conformers that differ only by one dihedral angle.

● A rotamer library is a collection of conformers for each residue type in proteins with side-chain degrees of freedom.

● It usually contains information on both conformation and frequency of a conformation.

Ponders & Richards, JMB, 1987 – analysis of internal packingDunbrack & Karplus, JMB, 1993 – application to side-chain predictionLovell et al., Proteins, 2000 – the penultimate rotamer library

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Protein design – work fow● Computational calculations of possible sequences for a desired structure or function

● Translate into DNA sequence and generate gene

● Produce protein and test it

● New calculations based on test results

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Design of a novel protein fold

Kuhlman et al. (2003) Science,302:1364

2D schematic of target fold (arrows define constraints):93 residue α/β protein with novel topology

3D models were generated by assembling 3- and 9-residue fragments from the PDB 172 →backbone-only models within 2-3Å RMSDs from each other.

For each model a sequence was designed using the RosettaDesign Monte Carlo search protocol & energy function. All amino acids except cysteine were allowed at 71 positions, the 22 surface β-sheet positions were restricted to polar AAs.

Simultaneous optimization of sequence and structure: Cycling between sequence design & backbone optimization (to identify lowest energy for a given sequence)

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Design of a novel protein fold

Kuhlman et al. (2003) Science,302:1364

Representatve part of Top7 in unbiased SAD densityComputatonal model 2.5 A x-ray structure

Comparison of computationally designed model and solved x-ray

structure (PDB 1QYS)

2D schematic of target fold (arrows define constraints):93 residue α/β protein with novel topology

Blue: designedRed: x-ray structBB RMSD = 1.17 Å

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Design of idealized proteins

Koga et al. (2012) Nature,491:222

Fundamental Rules for the connection of bb, ba, and ab

Emergent Rules for the connection of bba, abb, and bab

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Design of idealized proteins

Koga et al. (2012) Nature,491:222

NMRDesign superpositonRMSD

Rose

ta

Ener

gie

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Fold change through engineering

TIM- or (ba)8-barrelIGP synthase HisF

(PDB 1THF) 9-stranded barrelCheYHisF chimera

(PDB 3cwo)

Flavodoxin-like foldresponse regulator CheY

(PDB 1TMY)

Illegitmate reciombination

Bharat et al. (2008) PNAS,105:9942Eisenbeis et al. (2012) JACS,134:4019

8-stranded barrelCheYHisF chimera

(PDB 2lle)

Ciomputational design

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Computational receptor design

Changes in the binding pocket

wt ligand

protein

new ligand

A binding pocket is rearranged in order to recognize a new ligand

=> new therapeutics, vaccines, biosensors

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Computational enzyme design

Malisi et al. (2011) BIOspektrum,17:736

Substrate orTransiton state

Catalytc motf Structure databaseScafold

Designed enzymeShell 2: only fexibility

Shell 1: mutatons

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Computational enzyme design

Catalytc motf in new scafold Steric conficts (arrows)

Search space of possible mutants Soluton with conficts resolved

Malisi et al. (2011) BIOspektrum,17:736

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Outline● What we have learned from nature protein evolution→

– Permanence of protein in evolution● Reconstruction of evolutionary history

– Change of protein in evolution

● How can we applying the knowledge protein design→– Computational design– Design of proteins– Design of enzymes

5858

“Piecing together the evolution of proteins”

Alva et al. (2015) eLife

5959

Vocabulary of primordial peptides

Alva et al. (2015) eLife

6060

Vocabulary of primordial peptidesap28

Alva et al. (2015) eLife

TPR

RPS20

6161

Helical hairpin in TPR and RPs

TPR RPS20

Zhu et al. (2016) eLife

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From helical hairpin to TPR

M4N, a designed TPR from RPS20-hh

Zhu et al. (2016) eLife

6363

Origin of a folded protein from an intrinsically disordered ancestor

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Outline● What we have learned from nature protein evolution→

– Permanence of protein in evolution● Reconstruction of evolutionary history

– Change of protein in evolution

● How can we applying the knowledge protein design→– Computational design– Design of proteins– Design of enzymes