12864
`
`Biochemistry 1993, 32, 12864-12874
`
`Mechanism for the Rotamase Activity of FK506 Binding Protein from Molecular
`Dynamics Simulationst
`
`Modesto Orozco, I Julian Tirado-Rives, and \Villiam L. Jorgensen•
`
`Department of Chen1istry, Yale University, New Haven, Con11ecticu1 06511-8118
`Received June 4, 1993; ReviJed J.fanuscript Received Septe1nber I 3, J 993*
`
`ABSTRACT: Molecular dynamics (MD) and free energy perturbation (FEP) methods are used to study the
`binding and mechanism of isomcrization of a tetrapeptide (AcAAPFNMe) by FK506 binding protein
`(FKBP). Detailed structures are predicted for the complexes of FKBP with the peptide in both ground-state
`and transition-state forms. The results support a mechanism of catalysis by distortion, where a large
`number of nonbonded interactions act together to stabilize preferentially the twisted transition state. The
`two most important groups for the catalysis are suggested to be Trp39 and Asp31, but several other groups
`are identified as directly or indirectly involved in the binding and catalysis. However, the structural results
`do not support the notion that the keto oxygen of the immunosuppressive agents FK506 and rapamycin
`mimics the oxygen for the twisted peptide bond in the FKBP-transition-state complex.
`
`The isomerization of peptidic bonds is a difficult process
`with free energy barriers typically being 15-20 kcal/mot
`(Drankenberg & Forsen, 1971; Larive & Rabenstein, 1993).
`Most peptides correspond to secondary amides and are found
`exclusively in the trans conformation, which is about 2.5 kcal/
`mol more stable than the cis (Drankenberg & Forsen, 1971 ).
`However, the tertiary nitrogen in prolyl residues allows the
`coexistence of cis and trans isomers in proteins, which may
`require interconversion during protein folding. Considering
`the difficulty of this reaction, and the need for efficient folding
`mechanisms in vivo, the existence of enzymes which catalyze
`the isomerization of prolylpeptides (peptidylprolyl isomerases
`(PPls)) is not surprising. PPis were first characterized by
`Fischer and co-workers (Fischer et al., 1984), and since then
`different studies have demonstrated that these enzymes
`catalyze the cis....,.. trans ison1erism of prolylpeptides during
`the folding of various proteins (Fischer & Bang 1985,
`B~chinger, 1987; Lang et al., 1987; Jackson & Fersht, 1991).
`The interest in two PPis, FKBP (FK506 binding protein)
`and cyc!ophilin, increased dramatically after the discovery
`that they are receptors for the immunosuppressant agents
`FK506 (Harding et al., 1989) and cyclosporin (Fischer et al.,
`l 989a; Takahashi, et al., 1989). Particularly, FKBPwasfound
`to be the receptor for a family of immunosuppressant drugs
`which includes FK506, rapamycin, and ascomycin. It was
`demonstrated that the binding of the drugs to the protein
`yields con1plexes which inhibit T-cell activation (see Rosen
`& Schreiber, 1992).
`FKBP is a small, and highly conserved cytosolic protein,
`which was isolated and purified by Schreiber and co-workers
`(Harding et al., 1989), who also cloned the protein (Standaert
`et al., 1990). and determined the 3-D structure in solution
`(Michnick et al., 1991) and in the crystal (van Duyne et al.,
`1991). Actually, 3-D structures of the protein as well as of
`the protein bound to FK506 (van Duyne et al., 199la),
`rapamycin (van Duyne et al .. 1991b), and ascomycin (Mead(cid:173)
`ows et al., 1993) are available. The protein's structure features
`
`'This work was supported by NIH Grant 01132136 and a fellowship
`from the Fullbright Foundation,
`•To whom oorrespondence should be addressed.
`I On lelve from the Departmc:nt de Bioqufmic.a, Facultat de Qui mica,
`Universitat de Barcelona, 1fartf Franqu~ I, Bar«lona 08028, SPAIN.
`•Abstract published in Advance ACS Abstractr, November I, 1993.
`
`a five-stranded antiparallel f3 sheet and a short amphipathic
`a helix, which partially covers one face of the f3 sheet. The
`active site, defined as the region where the protein binds the
`drugs, is located between the a helix and the {3 sheet in a
`hydrophobic pocket for med by the side chains of Trps9, Tyr26,
`Phe45, Phe36, Phe~9, nes6, Tyrs2, and Va1ss.
`Both FK506 and rapamycin bind to FKBP as surrogates
`of prolylpeptides inhibiting the PPI activity (Schreiber, 1991;
`Rosen & Schreiber, 1992). The low Ki and the presence of
`a C9-keto oxygen orthogonal to the pipccolinyl ring suggest
`that both drugs interact with FKBP as n1imicsof the transition
`state for cis- trans isomerization. The fact thatcyclosporin,
`a potent imn1unosuppressant drug, was a powerful inhibitor
`of the PPI activity of cyclophilin led to the speculation that
`the immunosuppressant effects of these drugs were associated
`with their inhibition of PPI activity (see Rosen & Schreiber,
`1992). This attractive hypothesis was ruled out by two
`observations: (i) rapamycin and FK506 have very different
`mechanisms for immunosuppressant activity, although their
`inhibition of PPI activity is similar (Bierer et al., 1990a), and
`(ii) the analog 506BD is a strong inhibitor of FKBP but is not
`an in1munosuppressant (Bierer et al., 1990b). Accordingly,
`the relationship, if any, between the PP! and immunophilin
`activities of FKBP and cyclophilin ren1ains to be determined.
`The mechanisms for the PPI action of the immunophilins
`are also obscure. Fischer and co·workers (Fischer et al.,
`l989b) proposed a mechanism based on the formation of
`covalent adducts between the peptide and protein. Kofron et
`al. (1991) suggested an alternative mechanism in which the
`protonation of the proline nitrogen would facilitate the
`isomerization process. Other authors (Albers et al., 1990;
`Harrison & Stein, 1990; Park et at., 1992; Rosen & Schreiber,
`1992) have suggested a mechanism based on the preferential
`stabilization of the transition state by non bonded interactions
`(catalysis by distortion). However, the Jack of a structure for
`a complex between an im1nunophilin and a peptide has limited
`the experimental characterization of the rotamase mechanism.
`In this paper, we present a molecular dynamics (MD) study
`on the mechanism of peptide isomerization catalyzed by FKBP.
`Kinetic measurements have been made for els _...... trans
`isomerization of tetra peptides (Fischer et al., 1989; Albers et
`
`0006· 2960 /93/0432·12864104.00 /0
`
`© 1993 American Chemical Society
`
`Roxane Labs., Inc.
`Exhibit 1018
`Page 001
`
`

`
`Mechanism for Rotamase Activity
`
`Biochemistry, Vol. 31. No. 47, 1993 12865
`
`Table I: Force Field Parameters for the Ala3-Pro4 Peptide Bond•
`bond or angle
`equil. value
`force constant
`lOO
`C-NIV
`l.39
`C-NIV-CH
`5
`l 15.0
`C-NIV-CQ
`115.0
`114.0
`CH-NIV-CQ
`
`dihedral angle
`Ca(A3)-C(A3)-N(P4)-Ca(P4)
`O(A3)-C(A3)-N(P4)-Ca(P4)
`C(A3)-N(P4)-CD(P4)-Ca(P4)
`Ca(A3)-C(A3)-0(A3)-N(P4)
`
`equil. value
`120/0
`-"0/180
`-t20/180
`tS0/180
`
`force constant
`200
`200
`too
`lOO
`
`q (cis)
`q (Twhted)
`atom
`0.340
`Ca(Al)
`0.200
`0.437
`C(A3)
`0.500
`O(AJ)
`--0.457
`-0.500
`--0.570
`N(P4)
`--0.570
`Ca(P4), CD(P4)
`0.225
`0.285
`a The atom typeN\V correopondi to N(Pro4) of the twisted conformer;
`other atom types are from the standard A1iBER/OPLS force-field
`(Weiner et al., 1984; Jorgensen & Tirado-Rives, 1988), Distances are
`in A, angles in deg, chargei in electrons, and force oonstants in kcal/mo!
`Al, or kcal/mo\ rad 2, The dihe-Oral parameters are used a$ constrains
`to keep the cfs or rwfs1ed conformation. Standard AMBER torsional
`parameters for the Ala3-Pro4 bond are removed.
`
`twisted amide bond were derived from recent calculations on
`twisted dimethylacetamide (see Table I and Duffy et al.
`(1992)), These charges correctly reproduce properties of
`amides in solution, including free energies of hydration
`(Jorgensen & Tirado-Rives, 1988; Duffy et al., 1992).
`The crystal structure of FKBP bound to rapamycin (van
`Duyne et al., 199lb) was used as a starting point for the
`calculations. First, the peptide in the twisted form was overlaid
`on rapamycin. Theconforn1ation of the peptide was modified
`by rotation around single bonds to obtain the best fit with
`rapamycin and to reproduce well the environment of analogous
`hydrogen bond donor and acceptor groups. Once a suitable
`conformation of the peptide was obtained, the drug was
`removed, yielding a protein-peptide complex in which the
`peptide not only reproduces the general shape of rapamycin
`but also has a similar pattern of hydrogen bonds with the
`binding pocket.
`The protein-peptide model was solvatcd with a sphere of
`23·A radius containing TIP3P water (Jorgensen et al., 1983)
`centered on the center of mass of the peptide. This led, after
`removal of water molecules with unreasonably short contacts,
`to a salvation sphere with 1002 water molecules. A soft
`harmonic term with a force constant of 1.5 kcal/(mol A2) was
`used to retreive water molecules drifting beyond the boundary
`of the sphere. The resulting system is shown in Figure 2,
`where it is clear that the sphere of water provides extensive
`hydration of the binding site for the MD calculations. The
`solvated system was then divided into two regions: (i) an
`inner part, which includes the peptide, the water, and all
`residues which have at least one atom closer than 12 A to any
`atom of the peptide, and (ii) an outer part, which contains the
`rest of the protein. The first part includes 78 residues of the
`protein and was free to move in all minimizations and MD
`simulations, while the outer part with 25 residues was kept
`rigid in all calculations, This initial system was then prepared
`through 2000 steps of energy minimization (500 where the
`peptide and the protein were kept rigid, 500 where only the
`peptide was fixed, and 1000 where the entire system was
`minimized), and 40 ps of MD for heating from T == 198-298
`K and equilibration. The final equilibrated structure was
`used to run 500 ps of constant temperature MD at 298 K. The
`
`Pto.f
`Ala.3
`FIGURE l: Structure and nomenclature of the peptide used in the
`simulations.
`
`al., 1990i Harrison & Stein, 1990; Kofron et al., 1991; Harrison
`& Stein, 1992: Park et al., 1992). Consequently, structures
`for the complexes of a tetrapeptide with a cis peptide bond
`and of its transition state were sought and obtained here.
`Support for the model includes free energy results for mutation
`of the P 1 residue. Specific interactions responsible for selective
`stabilization of the transition state are identified. The model
`clarifies in detail the mechanism of rotamase action and is
`open to experimental testing, The binding insights should
`also be useful in the design of high-affinity ligands for FKBP
`with potential imn1unosuppressive activity.
`
`COMPUTATIONAL METHODS
`
`A peptide with the sequences Ace-Ala-Ala-Pro-Phe-Ame,
`where Ace (acetate) and Ame (methylamide) are the N- and
`C-terminal groups, was used to mimic the natural substrate.
`Figure 1 shows the structure and related nomenclature; in the
`following, plain numbering is used for the peptide residues
`and superscript numbering is used for protein residues. This
`sequence was selected because is resen1bles closely the synthetic
`peptides (Suc-Ala-X-Pro-Phe-pNA) used to nleasure the
`activity of PP!s (Fischer et al., 1989; Albers et al., 1990;
`Harrison & Stein, 1990; Kofron et al., 1991; Park et al., 1992).
`The peptide was built in both cis and twisted conformations
`around the Ala3-Pro4 peptidic bond, The peptide in the els
`conformation is used as a model of the substrate, and the
`twisted conformation, as a model for the transition state.
`The molecular dynamics calculations were carried out with
`the AMBER program, version 4.0 (Pearlman et al., 1991).
`The AMBER/OPLS force field (Weiner et al., 1984;
`Jorgensen & Tirado-Rives, 1988; Jorgensen & Severance,
`1990) was used to describe both the peptide and the protein
`interactions; the only modifications (see Table I) were for the
`peptidic bond Ala3-Pro4, where the torsional barrier and
`improper torsion at nitrogen were removed (in both planar
`and twisted conformations), and the bond lengths and angles
`at N(Pro4) were assigned to average values for planar and
`twisted amides (Duffy et al., 1992). The stretching and
`bending parameters related to N(Pro4) were reduced in order
`to allow the peptide to accommodate easily to the protein
`environment. Four constraints were used to guarantee that
`the Ala3-Pro4 peptidic bond stayed in the cis or twisted
`conformation. The same force constants were used for both
`conformers in order to make comparable the Hamiltonians
`for the els and twisted peptides (Table I). The contribution
`of these constraints to the total energy was not included in any
`of the simulations.
`Standard OPLS Lennard-Jones parameters were used for
`the cis and twisted structures (Jorgensen & Tirado-Rives,
`1988;Jorgensen & Severance, 1990). Standard OPLS charges
`for amides were also used to describe the planar structure
`(Jorgensen & Tirado-Rives, 1988), while the charges for the
`
`Roxane Labs., Inc.
`Exhibit 1018
`Page 002
`
`

`
`12866 Bioche111istry, Vol. 32, fi'o. 47, 1993
`
`Orozco ct al.
`
`\
`''
`
`FIGURE 3: Tl1crmodynarnic cycle used to determine the .6Li.G' of
`isomeri1ation lxtwcen the Ala and Gly peptide~. Dark lines arc the
`peptide and light lines arc !he r.ipamydn molecule.
`
`,\jN] . .'i.\
`
`?J.G = L -kTln (exp(-(V,i.,+r}~
`
`),~o
`
`V,..)/k'/1)~1
`
`(I)
`
`wide sampling windows of 5 ps each (2 ps of equilibration and
`3 ps of averaging) for a total of 205 ps for each mutation. The
`entire peptide was considered as the perturbed group in the
`n1utation; not only the intergroup interactions, but also all the
`intragroup nonbondcd interactions (including 1-·4) were
`considered in the evaluation of the free energy changes. \Ve
`had dctern1ined in pilot calculations th al the explicit inclusion
`of bonded contributions in the free energy calculation led to
`increased noise in the final free energies, but did not alter
`significantly the final results. The averaging in eq I was
`performed by lvt D simulations at constant T = 298 K. where
`T is the temperature, k is Bohz111ann's constant, ,\ is the
`coupling paramder, which controls the evolution or the Ala
`--.. Gly mutation, and V~ is the potential energy of the system.
`A similar simulation was performl'd to determine whether
`or not the structure of the ciJ peptide was able to c.xplain
`experimental binding data (Parker ct al., 1992). Thus, the
`difference in free energy of binding between Ala and Gly
`(cis)peptidcs (0.il.Gr.;r...i) was determined as the difference
`between the free energy changes in the mutation Ala-· .. G\y
`in water (l!.Gm~t"~ 1a), and in the protein comple.\ (~Gm~t''r«).
`In order to coniputc ilGmut"• 1n the Ala (cis)peptide in the
`conformation adopted inside FKBP at the end of the J\·ID was
`introduced into a cubic box (l'dgc ~~ 30 A) of TIPJP water.
`The closest water n10\cculcs wen.~ removed to yield toa system
`with 807 water molecules. The system was minimi1.ed for
`3000 cycles (2000 with the pl'ptide fixed and 1000 with the
`entire svstcm free). The systc1n was heated from 198 to 298
`K duri1;g 5 ps of NVT ~tD (during the first 3 ps only the
`water was allowed to n1ove), and equilibrated during 20 ps of
`NPT :0..tD (T = 298 K; P = I atm). The free cnergr change
`for the Ala - ~ Gly mutation was computed using 41 double(cid:173)
`widesampling windows of 2.5 ps ( 1.25 ps of equilibration and
`1.25 psof averaging) for a total of 102.5 ps. Periodic boundary
`conditions were used in all the simulations. All the re1naining
`technical details of the sin1ulations were identical to tho-;e
`noted above for the peptide-protein cmnplexc.s.
`
`RESULTS
`
`Transition-State S'rn1ctrtr1'. The strt1L'turc of the twisted
`transition state snndel. which w;is obtained after the fitting,
`npti1ni1ation, and c4uilibratio11 prot:css rcsl'rnbks the general
`
`F!GURP. 2: FKBP-pcptidc complex inside the 23-A sphcr.:: of water
`used in the l\1D simulations.
`
`integration step was 0.002 ps. and the coordinates were stored
`every 0.2 ps;. SJIAKE (Ryckacrl ct al., 1977) was used to
`maintain all bonds kngths al their cquilibriun1 values. A
`nonbonded spherical cutoff of 8 A and a residuc-b;1scd
`non bonded list, updated every 25 integration steps, were used.
`The structure at r = 100 ps. was used to generate the cis
`pcptide--protein complex. The change between 1wis1nl and
`cis peptides was done in si.x steps with the angles of the
`constraints and the charges gradually modified from fll'istcd
`to cis values (Table I). After each change, the peptide alone
`wa.;; optirni1.ed i111·an10 for 100 cycles; harntonic constraints
`were us~'d to guarantee that most of thcconfonnational changl'~
`would occur mainly in the Ala3--Pro4 urea. This si1nple
`procedure 1nimics a fast change from twisted to cis, and we
`found it to be superior to other 1nethods such as stepwise
`rotation for the full protein- peptide systen1 and si111ulated
`annealing.
`The cis peptide was then placed inside the binding pocket
`or the solvatcd protein occupying the san1c location as the
`1wisted conrorn1er. The S)S!Cm was opti1nized, hl'atcd, and
`equilibrated during 40 ps using a procedure analogous to that
`described above. A 500·ps !vtD trajectory was then run for
`It should be notl'd that the cis and twistt'd
`the cl-~ system.
`systems contain thcsa1ne number of ato1ns, identical definition
`oft he hydratinn shell, identical partitioning between the inner
`and outer regions, and, except for the charges, the san1e
`Ha1niltonian.
`Frceenergycalculations were used to predict the difference
`in isomerization rate between two peptides, Acc·Ala-Ala(cid:173)
`Pro· Phe·A1ne and Ace-Ala-Gly-Pro· Phe·Ame, for co1nparison
`with experi1nental data on the corresponding Suc-Ala-X-Pro·
`Phc-pNA peptides (Albers ct al., 1990; Harrison & Stein,
`1992; Park et al.. 1992). The thcr1nodyna1nic cycle in Figure
`3 was utilized to obtain the change inf rccenergiesof activation.
`The free energy changes for the AlaJ ____,,. GlyJ 1nutations was
`computed by statistical perturbation theory ac-cording to eq
`I (Zwa111.ig, l9S4). The.sc 1nutations were started with the
`structures and ve]qcities of the cis and !wisted systcnv~ at the
`end of the ~·1 D runs. The \;akulations were done in 41 double-
`
`Roxane Labs., Inc.
`Exhibit 1018
`Page 003
`
`

`
`Mechanism for Rotamase Activity
`
`Biochemistry, Vol. 32, No. 47, 1993 12867
`
`FIGURE 4: Superposition of the structures of bound rapamycin and the twisted peptide afler the e<J,uilibration.
`
`ALA3
`
`140.0
`
`340.0
`240.0
`l(ps)
`
`440.0
`
`540.0
`
`PHE5
`
`ALA2
`
`0
`
`" Vi
`
`120.0
`
`6-0.0
`
`·60.0
`
`·120.0
`
`-180.0 ~-~--~--~--~--~
`40.0
`140.0
`240.0
`340.0
`440.0
`540.0
`t(ps)
`
`PR04
`180.0 ~-~--~--~--~--~
`
`;1
`VJ
`
`120.0
`
`60.0
`
`0.0
`
`·60.0
`
`·120.0
`
`120.0
`
`60.0
`
`·60.0
`
`·120.0
`
`·60.0
`
`·120.0
`
`·180.0 ~-~~-~~-~--~--~
`40.0
`140.0
`240.0
`340.0
`440.0
`540.0
`l(ps)
`
`·t80.0 --~--~--~--~~---'
`140.0
`240.0
`340.0
`440.0
`540.0
`40.0
`l(ps)
`
`F!OURE 5: Dihedral angle histories for the twisted peptide.
`
`structure of rapamycin, as shown in Figure 4. The main chain
`of both molecules overlaps well, and the H·bond donors and
`acceptors are also in proximal positions. In particular, O(Ala3)
`is placed very close to the keto oxygen of rapamycin, and the
`Pro ring shares the space of the pipecolinyl ring.
`Analysis of the 500 ps trajectory shows flexibility in the
`peptide, especially for the side chain of Phe5 and in the Ala3-
`Pro4 region. The histories for the main chain dihedral angles
`shown in Figure 5 reveal that most movements are short range
`oscillations, which do not lead to dramatic changes in the
`structure. However, there is also a systematic movement
`
`occurring for the Ala3-Pro4 region in the 80-120 ps period,
`which leads to a significant change in the relative orientation
`of the peptide inside the protein, as shown in Figure 6, The
`conformation of the 60 and 540 ps structures are similar, as
`noted in Figures 5 and 6 with a general .. turn" topology
`stabilized by one or two intrachain hydrogen-bonds. The
`largest differences between the 60 ps and 540 ps structures
`are in the positioning of the praline ring and Ala3-Pro4 with
`respect to the binding site (see Figures 6 and 7). The reduction
`in Y, for Ala3 after 60 ps secures the hydrogen bonds for the
`turn structure and leads to better possitioning of the Ala3
`
`Roxane Labs., Inc.
`Exhibit 1018
`Page 004
`
`

`
`12868 Biochenlisrry, Vol. 32, 1Vo. 47, 1993
`
`Orozco et al.
`
`T\vistcd 60 ps
`
`O(Al:i.1)
`
`'l\vistcd 540 ps
`
`FIGURE 6: Representation of the structure adopted inside the binding site by rap..1111ycin. and the tw1:~1edpcptidc at 60 and 540 ps, A common
`reference axis was used to orient the molecules.
`
`Rapainycin
`
`I
`
`,
`
`,:!ftrtiu",:
`
`-
`
`'Ill
`
`L ~: TI<'l
`,;,
`
`_ rmn
`
`~ ~
`
`,
`
`F
`
`>
`
`~
`
`"
`
`FIGURE 7: The twisted peptide bound to the active site of FKHP.
`carbonyl group with respect to the Asp17 side chain (sec below).
`The distances for key interactions arc shown in Figure 8.
`A hydrogen bond exists between the Asp37 side chain and the
`an1ino groups of Ala2 and often Ala3, which arc proximal
`(Figures 6 and 7). Two intrachain hydrogen bonds arc also
`
`con1n1on: Arnc6-Acel, and Phe5-Ala2, which stabilize the
`turn structure. Another interesting interaction is the close
`contact (ca. 2.0 A) between the atnidc hydrogen of Phc5 and
`the N of Pro4. There arc no hydrogen bonds between the
`oxygen of AlaJ and the protein. The analysis of Figure 8
`
`Roxane Labs., Inc.
`Exhibit 1018
`Page 005
`
`

`
`:Vlcchanism for Rotamast: Activity
`
`1Jiochen1istry, Vol. 32, Nv. 47, l'J'J3
`
`12:-:69
`
`.
`
`6.0 ~. d{O(P<)·:;~I~))
`I
`~ 5.o
`g 4.0
`.~ 3.0
`u
`2.0
`
`1.0 -
`40.0
`
`360.0
`200.0
`l(p!.)
`
`520.0
`
`6
`
`5.0
`
`-U
`
`I
`
`i
`
`'
`i
`
`I
`
`•
`'
`
`d{0{3)--002(037))
`
`6.0
`
`I
`
`5.0
`~
`~ 4.0
`g
`~ 3.0
`2.0
`_______ .... __ , __ __._--...........L
`1.0
`520.0
`40.0
`200.0
`300.0
`l{ps)
`
`d{Hll{A2)-002{037))
`6.0 ~~---,·-·------,---------r
`
`1.0
`40.0
`
`60 r_01~C{A3)-00>(0J1))
`
`I
`
`'
`
`'
`
`__ 5.0
`~ '
`g 4.0
`
`~ 3.0
`u
`
`2.0
`
`1.0 - ·
`3f.i0.0
`40.0
`200.0
`t(ps)
`
`52'0.0
`
`60
`
`d(Htl(A3)-002(037tu)) •
`
`50
`
`I::, i~
`'0 ~ 0-~---- '
`
`40 0
`
`3$).0
`200.0
`1(ps)
`
`S?\l 0
`
`.0 '~d(H>l~(Amo)-O(A~))
`" ¢ 4.0
`~ 3.0
`~·: -~-•---~
`
`40 0
`
`-
`
`520.0
`
`--------~--
`520.0
`360 0
`200.0
`200.0
`300.0
`l(ps)
`1{ps)
`F1GCR1; 8: Histork-s for ~elected interatomic distnnrcs during the MD ~imulation for the twi.11n/ peptide.
`
`reveals that thc-1noverncnts during the 80·-120 ps period have
`two major consequences: first the hydrogen bond b1:tween
`llcl6 and Pro4, which ntimics one of the hydrogens bonds in
`the crystal structure of the FK DP- rapamycin co1npkx is lost,
`and second, the intrapeptide hydrogen bond between Amc6
`and Ace\ is forntcd. Two kss dramatic, but particularly
`significant changes, are in the distance,~ between the Asp 37
`side chain and the C"·=O moiety of Ala3.
`In the starting
`confoT1nation, one of the negatively charged oxygens of Aspn
`is about 3.6 A from ()(AlaJ_) and 4.0 A from C(AlaJ), while
`in the final conforination these distances arc around 4.7 and
`J.9 A. respectively. Thus, the conformational change in the
`80-120-ps period transforms a clearly bad charge-dipole
`contact into a n1uch 111orc favorable one. It is also notable
`that after the confonnational change, lhe position of the
`carbonyl oxygen of AIClJ is 111on• si111ilar lo that of the a111ide
`oxygen rather than the keto oxygt'll of rC1pa1nyci11.
`The movc1nent of the 0(Ala3) fron1 the starting to the final
`conforn1ation leads to a n1odcst reduction in the nun1bcr of
`aryl interactions (a contact O(Ala3)-H(aromatic) is con·
`sidercd as an aryl interaction when the Q ... H distance is less
`than 3.2 t\) between 0(Ala3) and lhc aro1natic side chains
`of Phcl6, l'he99 , Tyr16, and Tyr~2 frmn 2.6 to 2.0. Analysis
`of the hydration of the peptide used a gcontetrie definition of
`a hydrogen bond H'quiring an N ... 11 or Q ... J-1 distance less
`than 2.5 A, and a donor- hydrogen -acceptor angle between
`!20 and !80°.
`\Vith this definition, there arc initially 5.4
`waters hydrogen bonded to the peptide on average. Thi~
`number increases to 5.8 in the !a~t 240 ps of the trajectory.
`One water is bound to O(Ala2). 1.3 to O(Accl), and 1.5 to
`0(l'he5 ): however. the latter's intrapeptidc hydrogen bonding
`partner, the amino group of Amc-6, is hydrogen bonded to a
`water only 10<,{; of the tin1e. O(Pro4) is not solvent exposed
`in thestartingconfonnation where it is H·bonded to llc~ 1'. but
`in the final part of the trajectory (I DO-~ 540 ps) it is hydrogen
`bonded to an average of I, I water n1nleculcs. Finally, ()(Ala3)
`is in a purely hydrophobic place during the first 320 ps of the
`trajectory, but at this P'-lint, it captures a water molecule,
`which remains hydrogen bonded during 809(.of the remaining
`time as in Figure 7 (an averageof0.4 water is bound toO(AlaJ}
`when the complete trajectory is considered).
`
`0{/\l•l)
`
`Cis 5-10 ps
`FIGURE 9: Reprc.>cntation of the conformation adopted in<;idc the
`active site by the cis peptide. Only the structure at the end of the
`~to run is shown, since no major changes occur for the ds peptide
`during the ~imulation.
`
`The last step in the analysis of the protein-peptide
`interactions was identifying water bridges co1111ccting polar
`groups of the peptide and the protein. There is a water bridge
`between O(Phe5) and the carbonyl of Ulu 54 during 55o/r, of
`the time, 1nintkking another one of the hydrogen bonds in the
`FKilP-rapa1nycin structure. A water bridge is also detected
`43~{> of the time between O(Ala2) and llis~ 7 , and one exis1s
`40'J, of the time in the l 20--J20-ps period between O(Pro4)
`and Tyr 82 . The latter bridge is Josi in Lhc final part of the
`i\'1[), when the watt:r on ()(/\.la3) rnakcs a bridge Ill the
`hydroxyl group of Tyr~2 during 80',~, of the time (Figure 7).
`Ci.1 Co11fon11atio11. The d.t !Onn of the bound peptide also
`has a turn structure, with obvious similaritic .. ~ to the structures
`of rapan1ycin and the fll"iHcd peptide ( Fi1:ure 9). The position
`of the peptide in the binding site is very si1nilar to that of
`rapamycin, wi1h the proline ring replacing the plpet:olinyl
`ring. The t"l.ID provides significant sampling of the config·
`urational space of the peptide, but nosystcntalicchangcs occur,
`as evident in the distributions of rnain chain dihedral angles
`shown in Figure IO. Key interactions arc indicated in Figures
`11 and 12. Thus, the AspH side chain is again hydrogen
`bonded to the a1nino group of A!a2, and often to the a1nino
`group of AlaJ. I lydrogcn bondsa!so form ocasiunally between
`
`Roxane Labs., Inc.
`Exhibit 1018
`Page 006
`
`

`
`12870 Biochemistry, Vol. 32, No. 47, 1993
`
`ALA2
`180.0 ~--------------
`
`' .,
`
`VI
`
`120.0
`
`60.0
`
`-60.0
`
`·120.0
`
`Orozco et al.
`
`ALA3
`
`60.0
`
`' " VI
`
`0.0 f - - - - - - - - - - - - - -1
`
`·60.0
`
`-120.0
`
`·180.0 ~-~--~--~--~---'
`40.0
`140.0
`240,0
`340.0
`440.0
`540,0
`t(ps)
`
`-180.0 ~~~--~--~-~~--~
`40,0
`140.0
`240.0
`340.0
`440.0
`540.0
`t(ps)
`
`PR04
`
`PHE5
`180.0 ~----------~--~
`
`120.0
`
`60.0
`
`-60.0
`
`·120.0
`
`120.0
`
`60.0
`
`0.0
`
`·60.0
`
`-120.0
`
`' " v•
`
`-180.0 '----~--~-~--~--__,
`40,0
`140,0
`240.0
`340.0
`440.0
`540.0
`t(ps)
`FJOURE 10: Dihedral angle histories for the els peptide.
`
`-160.0 ~-~--~~-~-~---'
`40.0
`140.0
`240.0
`340.0
`440.0
`540.0
`t(ps)
`
`)
`
`..I -t
`
`FrOURE 11: The els peptide bcund to the active site of FKDP.
`NH( Ame) and O(Ala2) and between NH(Phe5) and O(Ala2).
`Other H-bonds (not shown in Figure 12) that are present
`some of the time are between NH(PheS) and the imidazole
`ring of His!7 and between NH(Ame) and O(Pro4). A
`hydrogen bond exists between O(Ala3) and the hydroxyl group
`of Tyr26 only during short periods of time, for instance in the
`160-180-ps period, but most of the time this H-bond is not
`present. It is very interesting to look at the relative positions
`of As pH with respect to the carbonyl group of Ala3; the OD2-
`0(Ala3) distance is around 3,3 A, and the OD2-C(Ala3)
`distance is about 4.0 A. These reveal a repulsive charge(cid:173)
`dipolecontact, which contrasts the favorable interaction found
`
`for the transition state. Intrapeptide hydrogen bonds that are
`present in the twisted conformation, but are not present in the
`els structure are between NH (Phe5) and N(Pro4) and between
`NH(Ame) and O(Ace).
`The carbonyl oxygen of Ala3 is near the aromaticsidechain
`of Phe~ and Tyr26 (Figure 11); the average number of aryl
`interactions is 1.2 for this oxygen, which is ca. 1 less than in
`the twisted conformation. The els peptide is well hydrated,
`with an average of 5.0 waters hydrogen bonded (0.4-0.8 lcss
`than in the twisted structure). A detailed analysis of the
`hydration shell shows that the oxygens of Ace and PheS are
`the best hydrated, with an average of 1.9 and 1.8 water
`
`Roxane Labs., Inc.
`Exhibit 1018
`Page 007
`
`

`
`Mechanism for Rotarnase Activity
`
`d{HN(F5)-0(Af))
`6.0 ~----~-~
`
`Biochemistry, Vol. 32, No. 47, 1993 12871
`
`d(HN(.A.2)-002(03?))
`
`d(HN(A3)-002{037})
`6.0 ~-~-~--~
`
`"'-4.0
`
`5.0 <
`.~ 3.0
`
`'ti 2,0
`
`1.0 ~-~--~-~
`40.0
`200.0
`380.0
`520.0
`t{ps)
`
`1 ·~0~.0--200~.o--300~·~"--,~20.o
`t{pa)
`
`d(0(3)-002(037))
`
`d(C{.A.3)-002(037})
`
`6.0
`
`5.0 < I 4.o
`
`~ 3,0
`2.0
`
`6.0
`
`5.0
`
`g
`~ 4.0
`.i 3.0
`
`2.0
`
`520.0
`
`d(HN(""")--OiA'))
`
`6.0
`
`5.0
`g
`§ •.o
`~ 3,0
`v 2.0
`
`6.0
`
`g
`
`ro
`
`.'ti 3.0
`'ti 2.0
`
`360.0
`200.0
`t{ps)
`
`1.0
`40.0
`
`360.0
`200.0
`t(p•)
`
`520.0
`
`1.0
`40.0
`
`360.0
`200.0
`1(p•)
`
`520.0
`
`F10URE 12: Histories for selected interatomic distances during the dynamics for the els peptide.
`
`molecules bound. The oxygens of Pro4 and Ala2 are less well
`solvated with 0.5 and 0,7 water molecule bound on average.
`The amino group of Ame is hydrogen bonded to a water only
`10% of the time. O(Ala3) which is hydrogen bonded to a
`water molecule in the last part of the MD for the twisted
`conformer, is not solvated in the els conformation. Three
`water bridges exist sporadically; the most common involve
`Glu" which has water bridges with O(Pro4) and O(PheS)
`during 31 % and 22% of the simulation, respectively. A water
`bridge between O(Ala2) and the imidazole ring of His'7 also
`exists 38% of the time during the first 200 ps, but it is rare
`in the remaining 340 ps. Finally, there are no water bridges
`involving TyrS2 and 0(Pro4) or O(Ala3), in contrast to the
`twisted conformer.
`Free Energy Calculations. The structures determined for
`both the els and twisted complexes seem reasonable from the
`viewpoints of stability, interactions, and function. Never·
`thcless, doubt remains about whether or not they are the real
`conformations. Comparison of total potential energies dem·
`onstrates that the twisted form is slightly lower in energy than
`the cis, in good agreement with the proposed stabilization of
`the transition state by the enzyme, but the difference (around
`7 kcal/mol) is not significant considering the statistical noise
`in the simulations (±70 kcal/mo!). Additional MD calcu(cid:173)
`lations, graphical manipulation, and simulated annealing
`procedures support the correctness of the structures, but it is
`obvious that more quantitative results are desirable. Though
`an e;ii;perimental 3-D structure of the FKBP-peptide complex
`is not available, attempts could be made to reproduce kinetic
`data for the isomerization catalyzed by the enzyme. One
`possibility was to compute the potential of mean force (PMF)
`for rotation a bout the Ala3-Pro4 peptidic bond, and to compare
`the 6G of this process with the observed AG of activation for
`the enzymatic reaction (AG'). Unfortunately, several at·
`tempted PMF calculations using holonomic constrains en·
`countered major technical difficulties. Development of
`alternative procedures for such perturbations is under con·
`sideration,
`However, some support can be sought by comparison of the
`relative AG* of peptides with different sequence. Park et al.
`( 1992) found a difference in keat favoring Ala over Gly at the
`
`Pl position in the Suc-Ala-X·Pro·Phe-pNA peptide. Their
`kcat ratio of 1200 (T = 278 K) translates to a lower 6G 1 for
`theAla(Pl) peptide by 3.9 kcal/mo!. Harrison &Stein ( 1992)
`reported a ke1.1/Km ratio for Ala and Gly peptides of 44 (T
`= 283 K), which considering the Km for these peptides (Park
`et al., 1992) translates to a lowering of AG' for the Ala(Pl)
`peptide by 2.7 kcal/mo!. Finally Albers et al. (1990) found
`a k.,. ratio between Ala(PI) and Giy(Pl) peptides of 12,
`which considering the Km values of Park ct al. (1992) yields
`a difference in AG' of 1.9 kcal/mo! (T = 278 K) favoring