Computational design of protein function
Loren Looger
Hellinga lab
1. Allowable structures for proteins, DNA, small molecules
Progesterone
2. Pseudo-geometric potential
H-bonds
electrostatics
sterics
solvation
Pretty much like CHARMM...
E
r
E=Ar12 −
Br6
E'(r)=E(rmin −rmin −rα
),r <rmin
~ 1.1
Hydrogen bonds, too...
D
HA
anchorr
5roptr
⎛
⎝ ⎜
⎞
⎠ ⎟
12
−6roptr
⎛
⎝ ⎜
⎞
⎠ ⎟
10
cos2θ cos2 φopt−φ( )-8 · { } · ·
Area-based solvation energy
P P
H H
H
P = polarH = hydrophobic
Electrostatic potential
€
E =q1q2
εr
is a function of atom-type pair &protein environment.
Parameterized to fit experimental data.
3. Algorithm for choosing best structure(s) from all available
Complementary Surface Construction:Complementary Surface Construction:
Molten zone
Evolving zone
Fixed zone
Ligandcoordinates
Proteincoordinates
Poly-alaninePCS
Rotationalligand
ensembleDocking
grid
Force field
Placedligand
ensemble
Fixed ligand ensemble
Side-chainrotamers
EvolvedPCS
ensemble
Ranked PCS ensemble
Experiments
Periplasmic Binding Protein (PBP) scaffolds
MetabolitesMetabolites
ExplosivesExplosives PollutantsPollutants
DrugsDrugsNeurotransmittersNeurotransmitters
Chemical ThreatsChemical Threats
NH
HO
NH3+
TNT RDX
MTBE
D-lactateL-lactate
5-fluorouracil
ibuprofen
PMPA~soman
serotonin
NH3+HO
HOdopamine
N
N
NNO2
-
NO2-
NO2-
NO2-
NO2-
CH3
NO2-
O
OH
O
CH3
CH3
CH3
CH3
CH3
O
H3C
CH3
CH3
CH3
NH
NH
O
O
F
CH3
H-OOC
H3C
CH3
H
O
CH3C
O
O
H
&
[L-lactate] (µM) 0
0.5
1
40 80
Fx
Kd = 2 µM
Fx
0
0.5
1
52.5 100
Kd = 6 µM
[serotonin] (µM)
0
0.5
1
12.5 25
Kd = 2 nMxF
[TNT] (nM)
0
0.5
1
0 150 300
Fx
Kd = 4 nM
[5-fluorouracil] (nM)
Fx
0
0.5
1
50 100
Kd = 6 µM
[MTBE] (µM) 0
0.5
1
0.25 0.5
Fx
Kd = 45 nM
[PMPA] (µM)
QSAR Results for binding affinities QSAR Results for binding affinities for L-lactate & TNT Receptorsfor L-lactate & TNT Receptors
Calculated affinities from
€
log K
d
( ) = c
+ c
Δ G
elec
+ c
A + c
4
N
unsat
+ c
5
N
clash
+ c
6
s − s
0.
linear regressi oncoefficients, c…c6,obtained by a least-square s f it of t he experimenta l data; ΔGelec electrostat ic contribution;A nonpolar contact area between receptor and ligand;Nunsat number of unsatisfied hydrogen bonds i nthe ligand;Nclash number of steric clas hes between t heli gandand receptor (defi neda s contacts >5 kca/l mo );l s rati o of the volumes of the wild-type li gandto t hetarge t ligand;s0 apparen toptimum value of s for a particular li .gand
-10
-8
-6
-4
-2
0
log Kd (obs) -8 -4-6 -2
QBP
GBP
ABP
HBP
1100µM
10
101
100mM
0.1
RBP
L-lactate designs
QBP
GBP
ABP
HBP
1100µM
10
101
100mM
0.1
RBP
The use of QSARs in the predictions improves the designs: D-lactate
Construction of biological sentinels for Construction of biological sentinels for chemical threats and pollutantschemical threats and pollutants
[inducer]
exp
ress
ion modulation binary
Unicellular sentinels for chemical threats and pollutants
- + - +
Ribose
Lactate
5 Fluoro-uracil
TNT
MTBE
100 M 10 M 1 M 0.1 M 0.01 M 0.001 M
IPTG 0 M TNT
[TNT]
100 M2,4-DNT
100 M2,6-DNT
Dose Response of TNT Signaling
-1
-0.5
0
0.5
1
0 10 20 30 40 50 60
Ab
sorb
ance
210n
m
-1
-0.5
0
0.5
1
-1.5
-1
-0.5
0
0.5
1
1.5
0 10 20 30 40 50 60
Fraction #
Wt Gbp
L-Lac.G1
D-Lac.G1
KdlactateD L
none none
200µM 3µM
0.8µM 10µM
Immobilized receptors
Racemic mix
Optically pure enantiomers
0 10 20 30 40 50 60
LD
L
D
Computational design of ligand-binding sitesStrategy #2: predefined geometries
{ l, 1, 2, 1, 2, 3 }n
geometrical description of
essential features in the
complementary surface
side-chain rotamer library
+...
Site 1
Site 2
Combinatorial search
(108 sequence1012 rotamers)
Calculation #1Initial placement of PCS
on scaffold backbone
Design scaffold coordinates
Pairwise of atomic interactions
Complementarysurface
construction(1010-10200
rotamers)
Site 1
Site 2
+...
Calculation #2Complementary
surface construction(PCS + SCS)
Triose phosphate isomerase chemistry
Acknowledgements
• Mary Dwyer
• Jeff Smith
• Shahir Rizk