Celik, Leyla

Helligsø Jensen, Henrik

Koldsø, Heidi

Schiøtt, Birgit

Severinsen, Kasper

Sinning, Steffen

Tran, Thuy Tien

Wiborg, Ove

Elsevier - Publisher Connector 

428a Tuesday, February 23, 2010Platform AM: Protein-Ligand Interactions
2210-Plat
Conformational Transitions Upon Ligand Binding: Holo Structure Predic-
tion from Apo Conformations
Daniel Seeliger, Bert L. de Groot.
MPI for biophysical chemistry, Goettingen, Germany.
Biological function of proteins is frequently associated with the formation of
complexes with small-molecule ligands. Experimental structure determination
of such complexes at atomic resolution, however, can be time-consuming and
costly. Computational methods for structure prediction of protein/ligand com-
plexes, particularly docking, are as yet restricted by their limited consideration
of receptor flexibility, rendering them not applicable for predicting protein/li-
gand complexes if large conformational changes of the receptor upon ligand
binding are involved. Accurate receptor models in the ligand-bound state
(holo structures), however, are a prerequisite for successful structure-based
drug design.
Hence, if only an unbound (apo) structure is available distinct from the ligand-
bound conformation, structure-based drug design is severely limited.
We present a method to predict the structure of protein/ligand complexes based
solely on the apo structure, the ligand and the radius of gyration of the holo
structure. The method is applied to ten cases in which proteins undergo struc-
tural rearrangements of up to 7.1 A backbone RMSD upon ligand binding. In all
cases, receptor models within 1.5 A backbone RMSD to the target were pre-
dicted and close-to-native ligand binding poses were obtained for eight of
ten cases in the top-ranked complex models.
The developed protocol is expected to enable structure modeling of protein/li-
gand complexes and structure-based drug design for cases where crystal struc-
tures of ligand-bound conformations are not available.
2211-Plat
Design and Characterization of Small Molecule Inhibitors of the PICK1
PDZ Domain with Binding Free Energy Calculations
Irina Moreira1, Xiaoxia Ge1, Kenneth L. Madsen2, Ulrik Gether2,
Harel Weinstein1.
1Weill Medical College of Cornell University, New York, NY, USA,
2University of Copenhagen, Copenhagen, Denmark.
PDZ domains are scaffolding proteins that assemble and regulate many cellular
signaling pathways by recognizing specific C-terminal type II peptide se-
quences. Consequently, these domains are associated as well with human dis-
ease and represent putative targets for new pharmacotherapeutics. The PICK1
(Protein Interacting with C Kinase 1) contains a N-terminal PDZ for which the
first small molecule inhibitor (FSC231) was identified from fluorescent polar-
ization assay screening in the lab of Ulrik Gether (Thorsen TS, Madsen KL, Re-
bola N, Rathje M, Anggono V, Bach A, Moreira IS, Stuhr-Hansen N, Dyhring
T, Peters D, Beuming T, Huganir R, Weinstein H, Mulle C, Strømgaard K,
Rønn LCB, Gether U - submitted). To identify the binding modes of FSC231
in both the wild type and a K83H mutant for which FSC231 exhibited higher
affinity, we undertook computational docking of the compound to the crystal
structure of the PDZ domain and subsequent refinement by Molecular Dynam-
ics simulations. Based on the structure of FSC231, a library of over 1000 novel
small molecules were designed by employing Ligbuilder GROW strategy and
were docked to the same PICK1 PDZ domain. The top-ranked molecules from
the docking results were subjected to binding free energy calculation using a po-
tential of mean force (PMF) simulation method (Woo and Roux, PNAS 2005)
with restraining potentials. This method samples the physical path of the pro-
tein-ligand binding, involving the decomposition of the binding process to sev-
eral stages, and had proven successful in predicting the binding affinity for a va-
riety of peptide-PDZ domains complexes. The results from the modeling and
free energy simulation work characterized the molecular interaction network
of PICK1 PDZ domain and guide the efficient rational design of new lead com-
pounds.
2212-Plat
Computational Design of Protein Interfaces with Receptor Flexibility
Samy Meroueh, Shide Liang, Liwei Li.
Indiana University School of Medicine, Indianapolis, IN, USA.
In this work, we computationally graft the binding epitope of various small pro-
teins obtained from the RCSB database to bind to barnase, lysozyme, and tryp-
sin using a previously derived and validated algorithm. In an effort to probe the
protein complexes in a realistic environment, all native and designer complexes
were subjected to a total of nearly 400 ns of explicit-solvent molecular dynam-
ics (MD) simulation. The MD data led to an unexpected observation: some of
the designer complexes were highly unstable and decomposed during the tra-
jectories. In contrast, the native and a number of designer complexes remainedconsistently stable. The unstable conformers provided us with a unique oppor-
tunity to define the structural and energetic factors that lead to unproductive
protein-protein complexes. To that end we used free energy calculations fol-
lowing the MM-PBSA approach to determine the role of non-polar effects,
electrostatics and entropy in binding. Remarkably, we found that a majority
of unstable complexes exhibited more favorable electrostatics than native or
stable designer complexes, suggesting that favorable electrostatic interactions
are not prerequisite for complex formation between proteins. However, non-po-
lar effects remained consistently more favorable in native and stable designer
complexes reinforcing the importance of hydrophobic effects in protein-protein
binding. While entropy systematically opposed binding in all cases, there was
no observed trend in the entropy difference between native and designer com-
plexes. A series of alanine scanning mutations of hot-spot residues at the inter-
face of native and designer complexes showed less than optimal contacts of
hot-spot residues with their surroundings in the unstable conformers, resulting
in more favorable entropy for these complexes. Finally, disorder predictions re-
vealed that secondary structures at the interface of unstable complexes ex-
hibited greater disorder than the stable complexes.
2213-Plat
The Two Enantiomers of Citalopram Bind to the Human Serotonin Trans-
porter in Reversed Orientations
Heidi Koldsø1, Kasper Severinsen2, Thuy Tien Tran1, Leyla Celik3,
Henrik Helligsø Jensen1, Ove Wiborg2, Steffen Sinning2, Birgit Schiøtt3.
1Department of Chemistry, Aarhus University, Aarhus C, Denmark,
2Laboratory of Molecular Neurobiology, Centre for Psychiatric Research,
Aarhus University Hospital, Risskov, Denmark, 3iNANO and inSPIN
Centers, Department of Chemistry, Aarhus University, Aarhus C, Denmark.
The human serotonin transporter exhibits substantial selectivity in the binding
affinities of the two enantiomeric forms of the antidepressant citalopram. Pre-
vious studies of the structural determinants of S- and R-citalopram binding re-
vealed residues Tyr95 and Ile172 as involved in the discrimination between the
two enantiomers. However, the overall orientation of the ligand in the binding
site and the precise nature of protein-ligand interaction remain unknown. In this
study the binding of S- and R- citalopram to a homology model of the human
serotonin transporter are extensively examined via Induced Fit Docking (IFD),
QM-polarized Ligand Docking (QPLD), and GRID calculations. This resulted
initially in two binding models for the R-enantiomer and two models for the S-
enantiomer; however, we were able to propose one for each enantiomer through
computational methods. These proposed binding modes were validated with
biochemical experiments using a large battery of twelve different human sero-
tonin transporter mutant proteins. These mutants in combination with six differ-
ent optically pure citalopram analogs were utilized in a Paired Mutant Ligand
Analogue Complementation study. By this strategy important protein-ligand
interaction points could be traced hereby validating a binding model for each
enantiomer by itself. This provided a detailed picture of how the high-affinity
serotonin selective reuptake inhibitor, S-citalopram, and its low-affinity enan-
tiomer occupy the binding site of human serotonin transporter. The results
show that the two enantiomers have contrasting orientations of the para-fluo-
rophenyl and 1,3-dihydroisobenzofuran-5-carbonitrile groups of citalopram.
In the validated model for S-citalopram the fluoro-group is located in close
proximity to the pocket lined by Ala173 and Thr439 and the cyano-group is
found close to Phe341 and vice-versa for the R-enantiomer
2214-Plat
T Cell Receptor Cross-Reactivity Directed by Antigen Dependent Tuning
of Peptide-MHC Molecular Flexibility
Brian M. Baker.
University of Notre Dame, Notre Dame, IN, USA.
T cell receptor recognition (TCR) of an antigenic peptide presented by a major
histocompatibility complex protein (MHC) is required for a cellular immune
response. TCRs are intrinsically cross-reactive, capable of recognizing multiple
peptide/MHC complexes. TCR cross-reactivity is necessary for the normal
functioning of the immune system, but has also been implicated in numerous
pathologies. Multiple mechanisms have been postulated for TCR cross-reactiv-
ity, including molecular mimicry, where ligands share crucial chemical and
structural features, and conformational adaptability, where flexibility allows re-
ceptors to ‘adapt’ to different ligands.
The human TCR A6 recognizes the Tax peptide derived from HTLV-I when
presented by the class I MHC HLA-A2. However, A6 also recognizes the
Tel1p peptide from S. cerevisia. To investigate how A6 cross-reacts between
these two ligands, we determined the structures of the A6-Tel1p/HLA-A2 ter-
nary complex, the unligated A6 TCR, and the unligated Tel1p/HLA-A2 com-
plex. The structures revealed that cross-reactivity occurs via conformational re-
arrangements on both sides of the interface, including the receptor, the peptide,


The Two Enantiomers of Citalopram Bind to the Human Serotonin Transporter in Reversed Orientations 

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The Two Enantiomers of Citalopram Bind to the Human Serotonin Transporter in Reversed Orientations

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