Mechanisms of hematin crystallization and inhibitionby the antimalarial drug chloroquineKaty N. Olafsona, Megan A. Ketchuma, Jeffrey D. Rimera,1, and Peter G. Vekilova,b,1

aDepartment of Chemical and Biomolecular Engineering, University of Houston, Houston, TX 77204; and bDepartment of Chemistry, University of Houston,Houston, TX 77204

Edited by Lara A. Estroff, Cornell University, Ithaca, NY, and accepted by the Editorial Board February 17, 2015 (received for review January 16, 2015)

Hematin crystallization is the primary mechanism of heme de-toxification in malaria parasites and the target of the quinolineclass of antimalarials. Despite numerous studies of malaria path-ophysiology, fundamental questions regarding hematin growthand inhibition remain. Among them are the identity of the crystal-lization medium in vivo, aqueous or organic; the mechanism ofcrystallization, classical or nonclassical; and whether quinolineantimalarials inhibit crystallization by sequestering hematin in thesolution, or by blocking surface sites crucial for growth. Here weuse time-resolved in situ atomic force microscopy (AFM) and showthat the lipid subphase in the parasite may be a preferred growthmedium. We provide, to our knowledge, the first evidence of themolecular mechanisms of hematin crystallization and inhibition bychloroquine, a common quinoline antimalarial drug. AFM obser-vations demonstrate that crystallization strictly follows a classicalmechanism wherein new crystal layers are generated by 2Dnucleation and grow by the attachment of solute molecules. Weidentify four classes of surface sites available for binding ofpotential drugs and propose respective mechanisms of drugaction. Further studies reveal that chloroquine inhibits hematincrystallization by binding to molecularly flat {100} surfaces. A 2-μMconcentration of chloroquine fully arrests layer generation andstep advancement, which is ∼104× less than hematin’s physiolog-ical concentration. Our results suggest that adsorption at specificgrowth sites may be a general mode of hemozoin growth inhibi-tion for the quinoline antimalarials. Because the atomic structuresof the identified sites are known, this insight could advance thefuture design and/or optimization of new antimalarials.

malaria parasites | heme detoxification | crystallization mechanisms |chloroquine | crystal growth inhibition

Whereas significant public health initiatives have eradicatedmalaria from North America, Europe, and other devel-

oped regions of the world (1), the disease remains endemic in theequatorial regions of Africa, South America, Southeast Asia, andOceania (2). Approximately 40% of the global population is atrisk for malaria infection, predominantly from the protozoanparasite Plasmodium falciparum (2). Very disturbingly, a re-surgence of the disease throughout the world has occurred sincethe 1960s due to the emergence and spread of Plasmodiumparasites resistant to chloroquine combination treatments (2, 3).Delayed parasite clearance has been recorded for even the mostrecent artemisinin-based therapies (4). The weak responses to thecommon antimalarial drugs underscore the urgent need for re-search into the critical processes of malaria parasite physiology.Malaria parasites residing in the erythrocytes catabolize he-

moglobin and release Fe(II) heme (5). The released heme rap-idly oxidizes to toxic Fe(III) hematin, which is sequestered ascrystalline hemozoin (6, 7). The traditional Western treatmentfor malaria, quinine, and its synthetic homologs (chloroquine,mefloquine, and others) (8–11) putatively works by blockinghematin crystallization (12). Available evidence suggests thatartemisinin, another antimalarial drug, binds to heme (2, 13).The sequestration of heme into hemozoin is a suitable target fornew antimalarials. Hence, an understanding of the mechanisms

of hematin crystallization and its inhibition by antimalarials mayprove to be influential for drug development (14). Despite manyyears of effort (7, 9, 12, 15–18), fundamental questions regardingthe mechanism of hematin crystallization and its inhibition re-main elusive. Among them are (i) What is the nature of theenvironment within the parasite where hemozoin crystals recruithematin and grow? The two likely candidates are the aqueousphase in the parasite digestive vacuole (DV) (18, 19) and thelipid subphase that has been reported to reside either in the DVbulk (9, 16) or along the DV membrane (18–20). (ii) What is themechanism of hematin crystallization?—classical, i.e., additionof molecules to growth sites (21–23), or nonclassical, i.e., asso-ciation of precursors (24–26)? (iii) What is the mechanism ofaction of the inhibitor species? It is possible that the inhibitorseither reduce the concentration and activity of hematin in thegrowth medium through complexation (27, 28), or interfere withcrystallization by binding to the crystal surface(s) and restrictingsolute addition (23).The answers to these questions offer an improved under-

standing of malaria parasite physiology and may potentially leadto the rational design of hematin crystallization inhibitors thatcould serve as effective antimalarial drugs. As a model of he-matin (Fig. 1A) crystallization we use the growth of β-hematin,the synthetic form of hemozoin. β-Hematin has a crystal struc-ture (P1 symmetry) and habit identical to its natural analog (7),with predominant growth along its ~c direction, (Fig. 1B). Bothnatural and synthetic hematin crystals assemble as high-aspect-ratio parallelogram-shaped platelets, with basal {100} faces andsides defined by {010}, (011), and (001) surfaces (17, 29).

Significance

Approximately 40% of the global population is at risk formalaria infection and 300–660 million clinical episodes ofPlasmodium falciparum malaria occur annually. During themalaria parasite lifecycle in human erythrocytes, heme re-leased during hemoglobin catabolism is detoxified by seques-tration into crystals. Many of the common antimalarials arebelieved to suppress the parasite by inhibiting hematin crys-tallization. We present, to our knowledge, the first evidence ofthe molecular mechanisms of hematin crystallization and an-timalarial drug action as crystal growth inhibitors. These find-ings enable the identification and optimization of functionalmoieties that bind to crystal surface sites, thus providingunique guidelines for the discovery of novel antimalarials tocombat increased parasite resistance to current drugs.

Author contributions: K.N.O., J.D.R., and P.G.V. designed research; K.N.O. and M.A.K.performed research; K.N.O., J.D.R., and P.G.V. analyzed data; and J.D.R. and P.G.V. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. L.A.E. is a guest editor invited by the EditorialBoard.1To whom correspondence may be addressed. Email: jrimer@central.uh.edu or vekilov@uh.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1501023112/-/DCSupplemental.

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Results and DiscussionAqueous or Organic Medium for Hematin Crystallization. The para-site DV presents a complex environment. The vacuole comprisesmembrane interfaces (20), an acidic aqueous solution with pH4.8−5.5 (30), and lipids, mostly mono- and diglycerides, resultingfrom the degradation of the transport vesicle membranes thatcarry hemoglobin into the DV (9, 16, 18–20). The location andstructure of the lipid subphase in the parasite DV is a subject ofdebate. Electron microscopy observations have indicated thatthe lipids self-assemble into nanospheres suspended in the DV(9, 16); however, recent studies indicate the absence of sus-pended lipid structures and suggest that the lipids line andthicken the DV membrane (18–20). Hemozoin crystals havebeen observed immersed in the lipid nanospheres (9, 16), or withtheir basal surfaces attached to the DV membrane (18–20) andother crystal faces apparently exposed to the aqueous subphase.Whereas it has been hypothesized that hemozoin crystals nucleateon the DV membrane (17, 20), the debate on the medium, aqueousor organic, from which soluble hematin reaches the crystals andassociates to them has yet to be reconciled (18–20, 31–33).To address this issue, we tested the feasibility of hematin

crystallization from solutions that mimic either the aqueousphase or the lipid structures in the DV. First, we attempted togrow hematin crystals larger than the hemozoin crystals extrac-ted from the DV of P. falciparum, which are less than 1 μm intheir longest dimension (16). Our group and others have pro-duced β-hematin crystals in aqueous solution, achieving a mar-ginal increase in crystal size (≤3 μm), but only through the use ofnonphysiological conditions (i.e., ionic strength 0.5–5 M) (34).Our attempts to grow β-hematin in aqueous solution using citricand acetate buffers (both at pH 4.8) as a surrogate for the DVproduced crystals with a morphology distinctly different fromthat of hemozoin (34). We then used an analog to the lipidsubphase in the DV, a solution of n-octanol saturated with citricbuffer at pH 4.8 (details of the preparation are provided in SIText) and referred to as citric buffer-saturated octanol, CBSO(35). In this solvent, we grew 30-μm β-hematin crystals thatpossessed the characteristic morphology (Fig. 1B) and powderX-ray diffraction pattern (Fig. S1C) of hemozoin crystals extractedfrom the DV of P. falciparum (17). Our results revealed that boththe organic and aqueous components are critical for crystal growth.For instance, β-hematin crystals failed to grow in anhydrousn-octanol, which seems to suggest that H+ ions are a necessarycomponent of the growth medium, presumably to facilitatethe formation of hydrogen bonds in the crystal structure (35).Our analysis of a representative blend of lipids in the DV suggeststhat there is ∼8.5% (by mass) dissolved water (Fig. S2).As a second test, we used in situ atomic force microscopy

(AFM) to monitor the evolution of unfinished layers on largeβ-hematin crystals in the presence of multiple aqueous solvents(Table S1). The unfinished layers did not grow despite theabundant growth sites presented on the curved steps (Fig. S3). Asimilar outcome was observed for anhydrous n-octanol (Fig. S4).However, this behavior is in direct contrast with the continuousgrowth of layers that were observed in CBSO solutions, as dis-cussed below. As a third test, we determined the solubility ofhematin in CBSO. Spectroscopic analyses (34) revealed that thissolubility is ∼105× higher than in aqueous buffer at pH 4.8 (Fig.S6A), which is not surprising given that hematin is a hydrophobicmolecule. Because crystal growth rates roughly scale with thesolubility (23), this disparity in the magnitude of hematin solu-bility indicates that crystallization from an organic phase is asignificantly faster method of heme detoxification than from anaqueous phase. Below, we use the data on hematin solubility forquantitative analyses of hematin crystallization in CBSO.An argument that is presented in the literature in favor of

aqueous crystallization of hematin is the putative low solubility

of the quinoline antimalarials in organic liquids, which wouldseemingly reduce their efficacy toward crystals growing in a lipidphase. To this end, we determined the solubility of chloroquine(CQ), a common antimalarial drug, in CBSO and citric buffer atpH 4.8 (Fig. S6B). The solubility of CQ in CBSO is 0.19 mM,which is ∼104× less than its solubility in the aqueous solvent, butit is still twofold higher than the solubility of hematin in CBSO(∼0.1 mM). Collectively, these measurements suggest that theCQ solubility in CBSO is sufficient for growth inhibition bysurface binding, a mechanism discussed in greater detail below.These arguments advocate that the lipid structures in the DV

may be a preferred environment for hematin crystallization.The difficulty in crystal growth from a physiologically relevantaqueous environment may be attributed to hematin’s low solubility(2 nM) (34) or its propensity to form oligomers (36, 37) that couldpotentially adhere to the crystal surface and slow or block itsgrowth. Even if one accepts that the crystals are not suspended inlipid nanospheres located in the DV bulk, but are attached to theDV membrane so that only one of the basal faces is exposed tolipids lining the DV membrane (18–20), this contact may be suf-ficient to ensure growth of the physiological hematin crystals.

The Crystallization Mechanism in CBSO. Guided by the conclusionof preferential β-hematin growth in organic solvents, we usedCBSO supersaturated with hematin as a growth medium andfocused on the {100} faces of β-hematin. We used large β-hematincrystals prepared in the biomimetic CBSO solutions discussedabove and performed, to our knowledge, the first time-resolved insitu AFM study of hematin crystal growth. In situ AFM has provento be a valuable technique for elucidating structural and dynamiccharacteristics of classical and nonclassical crystallization mecha-nisms (38–41). AFM topographical images (Fig. 1C) reveal thepresence of unfinished layers on a (100) face with heights h =1.17 ± 0.07 nm, close to the unit cell dimension in the [100]direction (a = 1.22 nm, Fig. 1D) (7).

Fig. 1. β-Hematin crystals. (A) Structure of hematin. (B) AFM image ofa β-hematin crystal on a glass substrate reveals a morphology similar tohemozoin crystals isolated from P. falciparum. (Scale bar, 2 μm.) (C) A 3DAFM height image of a (100) face reveals the presence of unfinished layers.The step height h = 1.17 ± 0.07 nm was determined by averaging mea-surements from multiple images. (D) Molecular model of β-hematin usingthe software package Diamond illustrates an unfinished layer (C atoms inwhite) on a (100) face (C atoms in blue).

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We observed a classical layer-by-layer mechanism whereinnew crystal layers nucleate and grow by the attachment of solutemolecules to advancing steps. Analyses of successive snapshotsfrom AFM movies reveal that new layers may either grow (I–IIIin Fig. 2 A–D), dissolve (IV in Fig. 2 A–D), or retain a steady sizeduring continuous imaging (V in Fig. 2 C and D) depending ontheir radius R (Movie S1). We observe a reduction in the criticalradius Rcrit for island growth or dissolution with increasing hematinconcentration (Fig. 2E), which is consistent with classical nucle-ation theory (CNT) applied to 2D crystal islands on a substrate(43). According to CNT, islands form as a result of fluctuations ofthe concentration of molecules on the surface. The dependenceRcrit(cH) is governed by the Gibbs–Thomson relation, according towhich Rcrit = Ωγ/kBTln(cH/ce) (23) [where Ω = 0.708 nm3 is thevolume of one molecule in the crystal (7); γ is the surface freeenergy of the layer edge; kB is the Boltzmann constant; T is tem-perature; cH is hematin concentration; and ce is hematin solubilityin CBSO]. The correspondence between the experimentally de-termined Rcrit and the a priori CNT prediction in Fig. 2F indicatesthat the generation of new layers on growing β-hematin surfaces isgoverned by the thermodynamics of hematin crystallization.Analysis of in situ AFM images permits the determination of

layer nucleation J2D as the number of islands that exceed Rcrit perunit area per time. According to CNT, J2D ∝ exp(–ΔGp

2D=kBT),where the free-energy barrier for layer nucleation, ΔGp

2D =πγRcrith, decreases with increasing cH, leading to an exponentialincrease of J2D with ln(cH /ce) (43, 44). Data in Fig. 2F arequalitatively consistent with this prediction, although the in-crease in J2D with ln(cH/ce) is weaker than this trend. This isexpected because J2D is regulated by surface supersaturationsthat are lowered from the bulk value during growth at high devia-tions from equilibrium, whereas Rcrit responds to surface super-saturations equilibrated with the bulk, as evidenced by the fluc-tuations of surface islands around their critical size in Fig. 2 A–D.

Upon nucleation, layers advance across the surface, mergewith adjacent islands, and eventually cover the entire face (Fig. 3A and B). The island morphology undergoes a temporal shiftfrom an isometric to an anisotropic shape that elongates alongthe c⇀ direction. The velocity v of advancing steps was determinedfrom the average displacement Δx of steps over time by thecomparison of successive AFM images, similar to those in Fig. 3A and B. We observed a faster step velocity along the c⇀ direction,consistent with high c/b aspect ratios of islands and bulk crystalhabit, which may be attributed to the differences in kink struc-ture (45) or density (46) along each step edge. Herein, we reportstep velocity in the dominant c⇀ direction.The step velocity v exhibits a linear dependence on hematin

concentration cH (Fig. 3C) if the steps are separated by morethan 150 nm. This linearity indicates that hematin moleculeaddition to β-hematin crystals is a first-order reversible processwhere v(cH) reaches zero at ce and becomes negative at cH < ce,denoting step retreat due to crystal dissolution. The estimate ofcrystal solubility ce = 0.16 mM from in situ AFM (Fig. 3C) is inexcellent agreement with bulk crystallization data (Fig. S5). Thecoefficient of proportionality between v and cH is referred to asthe step kinetic coefficient β defined from v = βΩ(cH – ce). Inturn, β is proportional to the effective first-order rate constant kfor association of molecules to the steps (β = a k, where a is themolecule size). From the data in Fig. 3C, β = 4.3 μm s-1and k ≅104 s−1 for steps moving in the c

⇀direction.

The observations in Figs. 2 and 3 indicate a strictly classicalmechanism of hematin crystallization, observed for numerousother solution-grown crystals (23, 45, 47, 48). Note that in ourAFM studies of growing crystal faces, we never detected theassociation of preassembled species, such as hematin oligomers,which would be indicative of nonclassical growth (24–26, 49).

Fig. 2. Generation of crystal layers. (A–D) Time-resolved in situ AFM imagesshowing growing and dissolving islands on a (100) face at cH = 0.25 mM. Arrowsindicate newly nucleated islands (I–V), islands that growwith time (I–III), an islandthat dissolves (IV), and an island that retains its size for the duration of obser-vation (V). (Scale bar, 125 nm.) (E) Dependence of the critical radius of 2D nucleiRcrit on ln(cH/ce); the solid line is the predicted trend based on the Gibbs–Thomsonrelation with surface free energy γ = 23 ± 5 mJ m−2 estimated using the Turnbullempirical rule (42), γ = 0.3

��ΔHo

cryst

��/Ω2/3, where ΔHo

cryst = -37 ± 8 kJ mol−1 is thecrystallization enthalpy, determined from the temperature dependence of he-matin solubility in CBSO (Fig. S5); dashed lines delineate deviations due to theerror in ΔHo

cryst and γ. (F) Rate of 2D nucleation of new layers J2D (i.e., thenumber of islands per unit time and area that nucleate and grow above Rcrit).The solid line is interpolated to guide the eye.

Fig. 3. Layer growth on β-hematin {100} surfaces. (A and B) Time-resolved insitu AFM images of layer growth in solution at cH = 0.28 mM. (Scale bar,250 nm.) (A) Stack of six layers slightly elongated along the c

⇀direction. (B) As

layers advance and merge with other layers, the anisotropy of their shapeincreases due to a faster step velocity v in the c

⇀direction relative to the b

⇀di-

rection, i.e., v{001}/v{010}= 2.5. (C) Step velocity in the [001] direction as a functionof hematin concentration cH. The solid line is a linear regression (R2 = 0.947).

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Concurrent with this observation, characterization of the ho-mogeneity of supersaturated hematin solutions in CBSO by dy-namic light scattering revealed the absence of any aggregates ofsize 1 nm and larger (35). This excludes the possibility of non-classical crystallization of hematin.The values of J2D and v in Figs. 2 and 3, respectively, may

provide insight into hematin crystallization in vivo. For instance,electron micrographs of hemozoin crystals in the parasite DVreveal that the crystals can reach thicknesses in the [100] di-rection of ∼100 nm within 20 h (9). Our in vitro assays suggestthat this approximate rate of crystallization occurs around cH =0.22 mM. Indeed, at this cH the step velocity in the c

⇀direction

v = 0.10 nm s−1 (Fig. 3C) and the corresponding density of steps(determined by the rate of nucleation of new layers J2D) ⟨l

−1⟩ ≅0.008 nm−1. The crystal growth rate is the product r = h⟨l−1⟩v ≅ 8 ×10−4 nm s−1; this product is independent of the direction of stepmotion (50). With this r, a crystal that is 100 nm thick in the [100]direction has grown for ∼18 h, which is comparable to that ofhemozoin in vivo. The closeness of the two time periods suggeststhat the measured layer nucleation rates and step velocities arephysiologically relevant. It also suggests that the hematin con-centration in vivo is close to 0.22 mM. This value is only slightlyhigher than the solubility of 0.16 mM and significantly lowerthan the potential maximum of 16 mM (i.e., the total hematingenerated in an average parasite DV) (9). These two compar-isons imply that hematin is incorporated into hemozoin crystalssoon after its release during hemoglobin catabolism. Thus,even a moderate delay in crystallization may induce a significantaccumulation of toxic hematin, leading to parasite eradicationfrom its human host.Direct AFM observation of nucleation of new layers and their

spreading by incorporation of molecules identified four classes ofsites on the surface of growing hematin crystals that are impor-tant for growth and constitute potential binding sites for anti-malarial drugs. These sites are illustrated in Fig. 4A for the {100}faces and their atomic structures are depicted in Fig. 4 B–G.Class 1 consists of molecularly flat surfaces that are typicallylocated between steps (Fig. 4B). Class 2 refers to nuclei of newlayers (Fig. 4C), which may comprise several molecules, and canpotentially exhibit structures that are distinct from those oflarger layers. Class 3 includes the kinks located along the steps(Fig. 4 D–F). There are four types of kinks, obtuse and acute,located on steps spreading the positive and negative b

⇀and

c⇀ directions. Finally, class 4 contains groups of closely spacedsteps that may host large inhibitor molecules capable of bridgingmultiple step edges–terraces, as illustrated in Fig. 4G.

The Mechanism of Action of CQ. The prevailing hypotheses regardingthe suppression of hemozoin formation collectively assume thatantimalarials increase cH in parasite DVs. This process can occurif drug molecules form noncrystallizable complexes with hematinin solution (i.e., hematin complexation) (51) and/or if the mol-ecules bind to hemozoin crystals and impede the addition ofsolute to growing steps (i.e., crystal growth inhibition) (17). Thelatter mechanism was proposed by Sullivan for quinoline drugs(52) and by Leiserowitz and coworkers for artemisinin (53). Todate, definitive evidence for antimalarial mode(s) of actionremains elusive. Here we identify the mechanism of β-hematincrystal growth inhibition by CQ. In situ AFM measurementsreveal that the addition of CQ to hematin growth solutions leadsto slower step growth, fewer 2D nuclei, and more rugged stepedges (Fig. 5 A–E and Movie S2). The impact of CQ at con-centrations cCQ = 0–2 μM on layer generation and step propa-gation is summarized in Fig. 5 F–H. There is an exponentialdecay in J2D with increasing cCQ that is accompanied by a mono-tonic decrease in v (Fig. 5 G and H). We observe a completesuppression of layer nucleation and step growth at cCQ = 2 μM.These observations are consistent with the hypothesis that CQ

impedes crystallization by binding to hematin crystal surfaces, sim-ilar to inhibition mechanisms established for many biogenic andsynthetic crystals in the literature (23, 54). Furthermore, the datasuggest that a complexation mechanism cannot fully account forthe reduced growth rates measured by in situ AFM. If, for ex-ample, we assume that CQ forms a 1:1 CQ–hematin complex,the free hematin concentration would decrease by 2 μM. Basedon the v(cH) dependence in Fig. 3D, this decrease would en-gender a negligible reduction in v. This interpretation, however,does not rule out a potential role of CQ–hematin complexes (51)in hematin growth suppression because AFM imaging cannotidentify the adsorbed inhibitor species.CQ could adsorb to any of the sites listed in Fig. 4. AFM

studies reveal a site specificity of CQ for {100} terraces. Theconcomitant suppression in J2D and v at cCQ = 2 μM and theappearance of protrusions along advancing steps (Fig. 5 D andE) are consistent with a step-pinning (stopper) mechanism (55),i.e., CQ molecules preferentially adsorb on the crystal surfaceand block step propagation. This mechanism assumes that in-hibitor surface coverage is governed by the dynamics of ad-sorption. If the separation between a pair of adsorbed inhibitors

Fig. 4. Four classes of sites on the surface of growing hematin crystals to whichinhibitor molecules may bind and arrest layer generation and step growth. (A)AFM image of a hematin crystal growing in CBSO. (Scale bar, 250 nm.) Numbersindicate respective classes. (A, 1) Molecularly flat segments of the growing face.(A, 2) Newly nucleated islands. (A, 3) Kinks along steps. (A, 4) Closely spacedsteps. (B–G) Illustrations of the atomic structures of the four classes of sites: (B)Flat surfaces; (C) Island nuclei; (D and E) Acute and obtuse kinks on stepsgrowing in the positive and negative ~b and~c directions, respectively. (F) Profileview of an acute kink. (G) Profile view of a pair of closely spaced steps. The greenspheres indicate modifiers that are physisorbed on crystal surface sites.

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is less than 2Rcrit, the adsorbates enforce a curvature at which theadvancing step is undersaturated and growth is arrested (55). Atintermediate inhibitor surface concentrations, step pinning pro-duces rugged steps (Fig. 5E, arrow) and impedes 2D layer nu-cleation. The significance of this mechanism is reflected in thesensitivity of crystallization to cCQ (Fig. 5 F–H). When cCQ drops to0.25 μM, both the generation and the growth of new layers proceedwith considerable rate. This high sensitivity may be a critical factorunderlying the increased resistance of P. falciparum to CQ (2).Specifically, resistant strains may have developed means to lowercCQ in the DV to levels that permit effective heme detoxification.

ConclusionsHere we present, to our knowledge, the first determination of themolecular mechanism of hematin crystallization. Our results pro-vide definitive evidence resolving several long-standing open ques-tions on hematin crystallization and heme detoxification. Thehematin solubility in organic and aqueous solvents and the mor-phology and dynamics of hematin crystal surfaces held in super-saturated solutions suggest that water-saturated amphiphilic organicsolvents are a preferred growth environment. Time-resolved in situAFM observations demonstrate that hematin crystallization followsa strictly classical mechanism of crystallization wherein new crystal

layers are generated by 2D nucleation and grow by the associationof molecules from the solution. These mechanistic details identifiedfour distinct classes of crystal surface sites that play crucial roles ingrowth and could be potentially blocked by crystallization inhibitorsto prevent heme detoxification. We provide direct evidence thatCQ adsorption on hematin crystal surfaces arrests heme de-toxification by suppressing surface growth at concentrations as lowas 2 μM. The mechanism of CQ drug action was conclusivelyidentified: CQ adsorbs on {100} terraces between hematingrowth steps and blocks step propagation. Collectively, thesefindings may engender a paradigm shift in the rational design ofantimalarial drugs wherein the identification of molecules withsite specificity for binding to hematin crystal surfaces providesboth a vital criterion and platform for experimental and com-putational drug screening.

ACKNOWLEDGMENTS. We thank David Sullivan, Paul Roepe, and LeslieLeiserowitz for insightful discussions on hematin crystallization and hema-tin–drug interaction. This work was supported by the National Institutes ofHealth through the Nanobiology Interdisciplinary Graduate TrainingProgram of the Gulf Coast Consortia for Quantitative Biomedical Sciences(Grant T32EB009379), National Science Foundation (Grant MCB-1244568),NASA (NNX14AD68G and NNX14AE79G), and The Welch Foundation(Grant E-1765).

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Fig. 5. CQ inhibition of β-hematin growth. (A–E ) Time-resolved in situ AFM images of a (100) face growing in solution at cH = 0.28 mM. (Scale bar,250 nm.) (A and B) In the absence of CQ, the surface consists of extensive flat terraces that are progressively populated with new layers. (C–E ) The in-troduction of 1 μM CQ at 20 min results in suppressed nucleation of new layers and corrugated step edges; both observations are consistent with a step-pinning mechanism. (F ) A decrease in the rate of 2D nucleation of new layers J2D relative to that in the absence of CQ, J2D,o, with increasing CQ con-centration. (Inset) Structure of CQ. (G) Effect of CQ on the step displacement Δx with increased imaging time in the presence of CQ, which is normalizedby the product of interstep distance l and relative supersaturation, σ = (cH – ce)/ce. (H) A decrease in step velocity v relative to that in the absence of CQ, vo,with increasing CQ concentration.

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