Communications to the Editor Bull. Korean Chem. Soc. 2001, Vol. 22, No. 3 253
Molecular Dynamics Simulations of a Low Oxygen Affinity Mutant Hemoglobin
Hyun-Won Kim： Chang-HyunLee,* Seunho Jung,§ andYoungdoWon*
Department ofChemistry, Hanyang University, Seoul133-791, Korea
‘Department of Biochemistryand Institue of BasicMedical Sciences and MedicalEngineeringResearch Institute, Yonsei UniversityWonju CollegeofMedicine, Wonju220-701, Korea
土Divisionof GeneralEducation, Pyon잉aekUniversity, Pyon잉aek, Kyungki-do450-701, Korea
§Department ofMicrobial Engineering, Konkuk University, Moj indong93-1, Kwangjin-Ku,Seoul143-701, Korea Received October 27, 2000
Keywords: Molecular dynamics, Recombinant hemoglobin, Structure simulation.
A recombinant(r)hemoglobin(Hb),a96Vlt Trp,hasbeen produced by usingEs이lerMhia coli expression plasmid in which synthetichumana and § -globingenesarecoexpress ed with the Es이ieri이iia coli methionine aminopeptidase gene.1 Theartificial hemoglobin haslowoxygen affinityand high cooperativityinoxygenbinding with theHill constant of 2.2-2.6, which compares to thevalueof3.0 for normalHb A. The carbonmonoxy formof rHb(a96Val tTrp) canbe easilyconverted toaligatedT-like quaternarystructure.
These functional properties provide an opportunity to develop a potential candidate for hemoglobin-based blood substitutes, and therehavebeenextensivestudies to charac
terizethe mutant hemoglobin byNMR spectroscopy, X-ray diffraction and molecular dynamics (MD) simulations.1,2In rHb(a96ValtTrp), a valine located inthe ai-§2 (and a
§i) subunitinterface is replaced by a bulky tryptophan. 1H NMRspectroscopy data indicated that the mutant hemoglo
binhad thetertiary and quaternary structuressimilarto those ofHb Ainboth thedeoxy and carbonmonoxyforms.
Prior to the X-ray crystallographic structure determina tion, preliminary MD simulations of the deoxy T-state rHb (a96ValtTrp) were undertaken using CHARMM22with the standardpolar hydrogenproteinpotential parameter set (PARAM19).3 The rHb model was based on the crystal structureof deoxy-HbA with both 96ValtransformedtoTrp.
All side chain angles of the tryptophan residue with more than 10% incidence in the rotamer library were considered as theinitial conformation.4 The sidechain angles,为 and x arelisted in Table1. Theorigin of thecoordinatesystem was setto themiddleoftheC§ ofthetwo a96 side chains. The protein model was hydrated with 103 TIP3P water mole
cules. The system is partitioned by 10A and 15 A radius spheres into reaction, buffer and reservoir regions for sto chasticboundary (SB)MD simulations. A stochasticsolvent boundary potential wasapplied to the 15A sphericalsurface and the Langevin equations of motion were integrated for the buffer region atoms. Thefinalconformationderived from the second rotamer was chosen as the mutant structure by considering bulkiness and symmetric arrangement of the tryptophan residues. Theresult showed thatthea96Trpresi dues were sufficiently separated and directed into the 아§
interface so as to introduce an additional hydrogen bond between the indole nitrogen ofa96Trp and the carboxylate sidechain of §99Asp. Theuniqueoxygen binding properties of rHb(a96ValtTrp) were explained with the additional hydrogen bond.
Thecrystallographicdata of rHb아96Vl t Trp) was obtain
edlaterat the 1.9 A resolution,2 which revealed that 아96Trp was ina conformation different from thatpredictedby the previousMD simulation. The indole side chain of the tryp
tophan is directedaway from theinterface and intothe cen tral cavity. Thereis no evidence for the hydrogen bondbet ween a96Trp and §99Asp in the crystallographic structure.
In the structure, the indole nitrogen makes water mediated hydrogenbonds with §101Glu instead. The water mediated hydrogen bondsare proposed as the structuralbasis for the lowoxygenaffinity of the mutant hemoglobin.
We have extended the molecular model of rHb(a96Val tTrp) and performed SBMD simulations. Here, we use CHARMM27 with the all hydrogen protein potential para meter set.5 Our model isbasedonthe deoxy-Hbcoordinates obtained from Protein DataBank (pdb2hhb).6 Considering Table 1. a96Trp side chain conformations of the SBMD simulation structures"
No. Rotamer Library - 15 A spherical SBMDb 20 A spherical SBMDc
아1 Trp96 아2 Trp96 아 Trp96 아2 Trp96
X1 X2 X1 X2 X1 X2 X1 X2 X1 X2
I -70.4 土 7.0 100.5± 18.2 -63.3 -176.3 -88.5 128.9 -178.6 -108.8 -160.0 -104.0
II 64.91± 3.0 -88.9土 5.3 92.3 -71.0 106.2 -78.7 -179.3 -132.5 -157.3 -68.0
III -177.3 土 7.9 -95.1± 7.6 -86.5 86.9 -174.2 -116.3 178.6 -119.5 178.7 -115.3
IV -179.5 土 3.4 87.5土 3.8 -118.0 89.5 -177.1 71.8 -153.0 -106.7 -178.7 -118.0
aDataare given indegree. bFrom Ref. 1. These werebased onthe dynamics average structures. c Angles are averaged fbr the dynamics frames from90ps to100ps with the 0.1ps interval.
254 Bull. Korean Chem. Soc. 2001, Vol. 22, No. 3 Communications to the Editor
hydrogenbondingfeasibility and exposure into the solvent, we determinetheprotonationstate oftitratable residues. All asparticacids and glutamicacidsare in thecarboxylateform and all lysineshave £ -NH3+. a20, a50,a58, a87, a89,占2, 063, )377,從,阳7 andQ143 Hishave 8-NH and a45,a72, a103, a112, a122, a116 and a117 His have £-NH. 0146 Histidines are doubly protonated. Four hememolecules are included. 221 TIP3P water moleculesare placedat the crys
tal water positions in the 2hhb structure. Hydrogens are placed by using theCHARMMHBUILDfunction.
The a96Val sidechain isreplaced by the Trpindole ring.
Withoutconsultingthe newly available crystallographic data, the conformation of the indole ring is built intothe structure basedonthe rotamerlibrary as in the previous MD simula tion. Fourpossible rotamer side chainanglesof Table 1are usedto generatethe initialcoordinates. EachrHb(a96Vl — Trp)model is briefly minimizedinenergy to remove abnor
mal straindue tothe addition ofthe tryptophan rings. The originofthe molecular coordinate is set to themiddle ofthe C3 of thetwo a96sidechains. The system is solvated with a water sphere of 20 A inradius and is partitioned with 16 A and20 A radius spheres to setthe stochastic boundary con
ditions.7 112 or 113 TIP3P water moleculesareintroduced in the solvation process. Coordinates of hydrogen atoms and the bulk water moleculesareoptimizedinthe solvated sys tem. The inside ofthe 16 A sphere is the reaction region,in which atoms move following the normalmoleculardyna
mics. Atoms in the buffer region between 16 and 20 A spheres run under the Langevin stochastic dynamics. The outside of the 20A radius sphere is the reservoir region, whereallatoms are fixedin position.
The leap-frog integratorofCHARMM is used to integrate both Langevin and normalequationsof motionwith thetime increment of 1.0fs.All hydrogenconnected bondlengths are fixed with the SHAKE method.8 The SBMD simulation is performed for 100ps with each rotamer system at 300 K.
Thecoordinate trajectoriessaved at every 100fsareanalyzed toyieldtheconformation of theindole sidechain of a96Trp as summarized in Table 1. In contrast to the previous MD simulation with the 15A SB, our simulations convergeinto the conformation with 力=180o and " =-110o with the fluctuation of 10oapproximately. The dimers tendto be sym
metric. The crystallographic data are aX1=171.6o,以％ = -114.9o, d2%1 =162.3o and 叫几=-114.9, which are most closelyreproducedin the simulationIII. The 100ps snapshot ofthe simulation III is showninFigure 1. The sidechains of the mutanttryptophanresidues extrude intothecentralcav ity. In allsimulationtrajectories,we find one to threecrystal lographic orbulk water molecules located less than 4.0 A awayfromthe 97Trp indolenitrogen atom. Inthe 100ps tra
jectories, crystallographic and bulkwater molecules move about 3.5Aand 6 A in average, respectively. Some water moleculesare displacedasmuchas 18 A. The current simu
lation reproduces the conformation of the mutant residue and indicates the water mediated hydrogen bonds of the crystallographic structure.
Although thepreviousMD simulation failedtopredictthe
Figure 1. The central cavity of the mutant hemoglobin rHb (a96Val t Trp) is shown in the 20 A radius spherical stochastic boundary. The structure is the 100ps snapshot of the simulation III, which is very close to the crystallographic data. Hemes of subunits and a96W residues are also depicted in the figure. The tryptophan sidechains extrude into the central cavity.
conformation of themutantresidue,itwasa pristineattempt to explore the modified structure resulting from the site- directed mutagenesis. As we review the previous work, we find that the system size is not large enough to provide appropriate conformational space for the relatively large tryptophan residues and too many water molecules are addedintothe void as the bulk water. Here,wehave demon
strated that careful SBMD simulations with explicitsolvent watermolecules are able to predict the structural detail of proteinsproduced by the site-directedmutagenesis.
Acknowledgment. This work has been supported by the Brain Korea 21 Project, the Korea Research Foundation, Yonsei University and the Cray University Research and Development Grant. Thefacility of the KORDIC supercom puting centerwasextensivelyused to run the simulationcode.
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