Download Structural characterization of an antibody in complex with

CASE STUDY
Structural Characterization of
an Antibody in Complex with a
Serine Protease
Tina Yeh, PhD
Lead Scientist - Accelrys
Structural stabilization due to monoclonal antibody binding
influences the protease activity of human kallikrein-3
• Accurate protein-protein complex structure prediction and refinement with ZDOCK and RDOCK protocols
Since the advent of monoclonal antibody (mAb) technologies in recent years,
• CHARMm based Calculate
Mutation Energy protocol
predicts antibody mutants
with enhanced stability
of each chain contains three regions of sequence hypervariability, called the CDRs
numerous therapeutic mAbs have been developed into beneficial agents for the
treatment of a variety of human diseases. Antibodies are multi-domain structures
typically composed of two light chains and two heavy chains. The variable domain
(complementarity-determining regions). These versatile hypervariable regions are
responsible for the antigen binding affinity and specificity.
Proteases are widely distributed in nature
Asp-Ser catalytic triad of serine proteases.
and various deregulated protease activities
PSA is a well known biomarker for prostate
are frequently implicated in pathological
cancer, and implicated in prostate cancer
conditions such as cancer, cardiovascular
development and progression. However
and inflammatory disorders. The challenge
the exact role of its protease activity in
in developing an inhibitor for a particular
prostate cancer is still unknown. Recently the
protease is to achieve specificity, since
crystal structure of kallikrein-3 in complex
members of the same protease family
with an activating antibody has been
often have largely conserved binding sites.
solved (Menez et al, 2008), which affords
The highly specific nature of antibody-
insight into the structural determinants
antigen recognition makes it desirable
of recognition and sheds light on the
to consider designing antibodies as
nature of antibody-PSA interactions.
potential modifiers of protease functions.
Prostate-specific Antigen (PSA), also known
as kallikrein-3, is an androgen-regulated
serine protease expressed in prostate
tissue and shares the characteristic His-
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CASE STUDY: Discovery STudio
Method
The antibody 8G8F5 recognizes the binding epitope on the
Protein docking studies were performed in Discovery Studio
using the Dock Proteins protocols. The ZDOCK protocol performs
the initial global, systematic search of the orientations of the two
protein partners. Typically the larger protein (the receptor protein)
is kept fixed while moving the smaller protein (the ligand protein)
around the receptor protein. ZDOCK uses a grid-based rigidbody docking search in six dimensions utilizing the Fast Fourier
Transform (FFT) technique for efficiency. The rotational search
sampling grid can use a 15 degree grid which samples a total
of 3600 docked poses, or a 6 degree grid which samples a total
antigen PSA located at five discontinuous surface loop segments
including residues: 90-95, 98-101, 124-129, 175-179, 232-240
(see Figure 2). The antibody does not reach into the PSA active
site; rather it binds at the peripheral side of the active site cleft.
The heavy chain CDR loops contribute more to the binding
interaction than do the light chain CDR loops. All 3 heavy
chain CDR loop residues H:27-33, H:52-58, H:96-100C are in the
central core region of the binding interface whereas the light
chain CDR1 loop residues L:27D-32 and CDR3 loop residues
L:91-96 residues are involved in the peripheral binding area.
of 54,000 poses for more accurate results. The ZDOCK internal
scoring algorithm is based on a pairwise shape complementarity
function (PSC) and optionally delsolvation and electrostatic
terms can also be included. As part of the ZDOCK protocol, the
ZRANK function is used to rerank the docked poses. ZRANK is an
optimized energy scoring function based on weighted energy
terms of van der Waals, electrostatics, and desolvation. The RDOCK
protocol can be used subsequently for further refinement of the
docked poses, using a CHARMm-based energy minimization
scheme for the optimization of intermolecular interactions.
PSA and the antibody Fab 8G8F5 structures are taken from PDB
2ZCH. The ZDOCK protocol with a 6 degree rotational sampling
grid was used and several antibody CDR loop residues were
specified for filtering poses. Docked poses are ranked with ZRANK
and further optimized with RDOCK. The best pose is very similar
to the crystal structure of 2ZCH (Figure 1), demonstrating that
the ZRANK scoring function and RDOCK refinement successfully
discriminate the near native protein complex structure.
Figure 2. Two views of the PSA-antibody binding interface are shown as ribbon
diagrams. Antibody heavy chain (green) and light chain (orange). The antigenic
determinants on PSA include five discontinuous segments: three loops (purple)
[90-95] [98-101] [175-179], one loop [124-129] (yellow) and the C-terminal helical
region [232-240] (yellow). The PSA catalytic triad residues H57 D102 S195 are
labeled.
As shown in the figure above, the three PSA loops [90-95] [98-101]
[175-179] are all in close spatial proximity and interact with all
three of the antibody’s heavy chain CDRs. The so-called classic
“Kallikrein loop” in PSA includes an 11 amino acids insertion
95A-95K (relative to standard chymotrypsin numbering). The
kallikrein loop in PSA is located between loop [90-95] and
loop [98-101], at the border of the PSA active site cleft.
PSA:His91 forms a hydrogen bond with antibody heavy
chain CDR3 residue H:Tyr97, and PSA:Pro92 forms a hydrogen
bond with heavy chain CDR2 residue H:Ser54. PSA:Leu93
is located in a hydrophobic pocket with favorable van der
Waals interaction with surrounding antibody heavy chain CDR
residues including H:Tyr33, H:Ala52, H:Pro52A, and H:Tyr97.
Figure 1. Protein ribbon diagram of the best docking prediction of the PSAantibody complex superimposed on the crystal structure from PDB 2ZCH. Antibody
light chain (orange), heavy chain (green).
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CASE STUDY: Discovery STudio
The PSA:Asp98 side chain can form hydrogen bonds with
antibody residue H:Thr28 or H:Thr31. At one edge of the
binding interface the PSA loop residue PSA:Lys175 also forms
a hydrogen bond with antibody residue H:Thr28. Another
PSA lysine residue PSA:Lys178 is within hydrogen bond
distance with antibody residues H:Asp96 and H:Tyr97.
The last segment PSA:232-240 is part of the PSA C-terminal helix
and interacts with the CDR loops from both the antibody light
chain and the heavy chain. At the edge of the binding interface
PSA:Lys236 can form a hydrogen bond with antibody residue
L:Ser91 and a salt bridge interaction with H:Glu100C. PSA:Arg235
forms a hydrogen bond with antibody residue L:Asp28.
With the solvent surface displayed on PSA, it’s easy to visualize the
different areas where each antibody CDR loop residue interacts
with PSA (see Figure 3). The heavy chain CDR3 loop occupies the
core region of the binding interface. The side chain of the two
lysine residues on PSA, Lys178 and Lys236, delineate a concave
shaped pocket on PSA that the heavy chain CDR3 loop fits
into. Another pair of lysine residues on PSA, Lys178 and Lys175,
Figure 3. Four lysine residues involved in the binding interface. PSA is represented
as a surface with the four lysine residues (Blue) labeled. Ribbon diagram for the
three heavy chain CDR loops displayed in yellow, with CDR3 loop in the center of
the screen (between Lys236 and Lys178). Ribbon diagram for the light chain CDR1
and CDR3 loops displayed in red.
delineate another pocket that the heavy chain CDR1 loop fits into.
At the edge of the binding interface, PSA residue Lys239 interacts
favorably with antibody light chain CDR1 loop residue L:Phe27D.
Several hydrophobic and aromatic amino acids from the
antibody heavy chain CDR3 loop and from PSA contribute to
the stabilizing hydrophobic core and favorable π interactions
in the binding interface. These include residues: H:Tyr32,
H:Tyr33, H:Tyr58, H:Tyr97, H:Phe99, and PSA:Leu93, PSA:His91,
PSA:His101, PSA:Phe179, PSA:Tyr234, PSA:Trp237.
Since PSA, also known as kallikrein-3, has a very long kallikrein
loop including the 11 residue insertion (from Met95A to Pro95K),
the flexible loop conformation could block the access to the PSA
active site nearby. Based on the allosteric regulation (activation)
mechanism, it has been proposed (Menez et al, 2008) that, the
antibody interacts with the two PSA surface loops [90-95] and
[98-101] on either side of the kallikrein loop, thereby effectively
stabilizing the kallikrein loop conformation and keeping the
substrate-binding cleft accessible. This could explain the
enhanced PSA enzyme activity upon antibody binding.
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We have performed computational scanning mutagenesis
analysis using the CHARMm-based Calculate Mutation Energy
protocols in Discovery Studio, to evaluate the effect of singlepoint mutations on the stability and binding affinity of the
PSA-antibody complex. For the Mutation Energy (Stability)
calculation, the energy effect of each mutation is calculated as
the difference between the folding free energy of the mutated
structure and the wild type structure. The method includes the
Generalized Born implicit solvent model in CHARMm and the
energy functional contains empirically scaled contributions
of van der Waals and electrostatic terms, a side chain entropy
term and a non-polar solvation energy term. The results have
identified several mutations on the antibody heavy chain that
exhibit a stabilizing effect. These include residue H:Ser54 on
the heavy chain CDR2 loop mutated to hydrophobic amino
acids TRP, TYR, PHE, ILE, and LEU, all stabilizing mutations, with
favorable contribution from the van der Waals energy term.
Residue H:Thr31 on the heavy chain CDR1 loop mutated
to MET, ILE, PHE, and VAL are also stabilizing mutations.
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CASE STUDY: Discovery Studio
Conclusion
References
In this case study we demonstrate the structural analysis
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of the binding interface of a PSA-antibody complex
using the tools and methods in Discovery Studio.
Protein docking studies using ZDOCK and RDOCK refinement
has successfully predicted the near native structure for the
PSA-antibody complex as determined by X-ray crystallography.
All three CDR loops of the antibody heavy chain contribute
the major part of the interaction with PSA, with the CDR3
loop at the core of the binding interface. The antibody light
chain CDR1 and CDR3 loops provide interaction at the edge
Menez et al, “Crystal Structure of a Ternary Complex between Human Prostate-specific Antigen, Its Substrate Acyl Intermediate and an Activating Antibody,” J. Mol. Biol. 2008, 376, 1021-1033.
LeBeau et al, “Prostate-specific antigen: an overlooked candidate for targeted treatment and selective imaging of prostate cancer,” Biol. Chem. 2010, 391, 333-343
Wu et al, “Structural insight into distinct mechanisms of protease inhibition
by antibodies,” PNAS 2007, 104(50), 19784-19789
Hedstrom L. “Serine Protease Mechanism and Specificity,” Chem. Rev. 2002, 102, 4501-4523.
Pierce B., Weng Z. “A Combination of Rescoring and Refinement Significantly Improves Protein Docking Performance,” Proteins 2008,
72, 270-279.
of the binding interface. The antibody-PSA binding affinity is
achieved through the shape complementarity between the
antibody paratope and the epitope on PSA consisting of five
discontinuous segments, the extensive hydrogen bonds, and
favorable van der Waals and hydrophobic interactions. The
computational scanning mutagenesis study has identified
a few putative mutations on the antibody heavy chain that
could potentially enhance the stability of the complex.
To learn more about Discovery Studio by Accelrys, go to
accelrys.com/discovery-studio
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