Search

Toxoplasma gondii is a widespread apicomplexan parasite that can cause severe disease in its human hosts. The ability of T. gondii and other apicomplexan parasites to invade into, egress from, and move between cells of the hosts they infect is critical to parasite virulence and disease progression. An unusual and highly conserved parasite myosin motor (TgMyoA) plays a central role in T. gondii motility. The goal of this work was to determine whether the parasite's motility and lytic cycle can be disrupted through pharmacological inhibition of TgMyoA, as an approach to altering disease progression in vivo. To this end, we first sought to identify inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant motor's actin-activated ATPase activity. The top hit to emerge from the screen, KNX-002, inhibited TgMyoA with little to no effect on any of the vertebrate myosins tested. KNX-002 was also active against parasites, inhibiting parasite motility and growth in culture in a dose-dependent manner. We used chemical mutagenesis, selection in KNX-002, and targeted sequencing to identify a mutation in TgMyoA (T130A) that renders the recombinant motor less sensitive to compound. Compared to wild-type parasites, parasites expressing the T130A mutation showed reduced sensitivity to KNX-002 in motility and growth assays, confirming TgMyoA as a biologically relevant target of KNX-002. Finally, we present evidence that KNX-002 can slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. Taken together, these data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target in infections with T. gondii. Since TgMyoA is essential for virulence, conserved in apicomplexan parasites, and distinctly different from the myosins found in humans, pharmacological inhibition of MyoA offers a promising new approach to treating the devastating diseases caused by T. gondii and other apicomplexan parasites. Toxoplasma gondii is a widespread apicomplexan parasite that can cause severe disease in its human hosts. This study shows that a small-molecule inhibitor of Toxoplasma myosin A reduces parasite motility and alters disease progression in vivo, suggesting a new approach to treating infections by apicomplexan parasites. [ABSTRACT FROM AUTHOR]

Trypanosoma brucei spp. develop into mammalian-infectious metacyclic trypomastigotes inside tsetse salivary glands. Besides acquiring a variant surface glycoprotein (VSG) coat, little is known about the metacyclic expression of invariant surface antigens. Proteomic analyses of saliva from T. brucei-infected flies identified, in addition to VSG and Brucei Alanine-Rich Protein (BARP) peptides, a family of GPI-anchored surface proteins herein named as Metacyclic Invariant Surface Proteins (MISP) because of its predominant expression on the surface of metacyclic trypomastigotes. The MISP family is encoded by five paralog genes with >80% protein identity, which are exclusively expressed by salivary gland stages of the parasite and peak in metacyclic stage, as shown by confocal microscopy and immuno-high resolution scanning electron microscopy. Crystallographic analysis of a MISP isoform (MISP360) and a high confidence model of BARP revealed a triple helical bundle architecture commonly found in other trypanosome surface proteins. Molecular modelling combined with live fluorescent microscopy suggests that MISP N-termini are potentially extended above the metacyclic VSG coat, and thus could be tested as a transmission-blocking vaccine target. However, vaccination with recombinant MISP360 isoform did not protect mice against a T. brucei infectious tsetse bite. Lastly, both CRISPR-Cas9-driven knock out and RNAi knock down of all MISP paralogues suggest they are not essential for parasite development in the tsetse vector. We suggest MISP may be relevant during trypanosome transmission or establishment in the vertebrate's skin. Author summary: The Trypanosoma brucei group of parasites are exclusively transmitted to the vertebrate host by the tsetse vector alongside insect saliva. To better understand trypanosome transmission, we investigated the protein composition of T. brucei-infected tsetse saliva using a mass spectrometry proteomics approach. We found that, in addition to proteins from tsetse saliva and Sodalis glossinidius (a bacterial tsetse symbiont), trypanosome-infected saliva contains several parasite surface glycoproteins, including a partially characterized family of invariant proteins herein named Metacyclic Invariant Surface Proteins (MISP). We show that MISP is primarily expressed, together with mVSG and BARP, on the surface of the infectious metacyclic stage of T. brucei. Its triple helix bundle architecture appears tethered to the outer membrane by an extended GPI-anchored C-terminal tail that putatively projects MISP above the VSG coat. Our findings provide new insights into the surface architecture of the T. brucei metacyclic stage and describes the challenges associated with developing transmission-blocking vaccines against tsetse-transmitted trypanosomes. [ABSTRACT FROM AUTHOR]

Toxoplasmosis is a neglected parasitic disease necessitating public health control. Host cell invasion by Toxoplasma occurs at different stages of the parasite’s life cycle and is crucial for survival and establishment of infection. In tachyzoites, which are responsible for acute toxoplasmosis, invasion involves the formation of a molecular bridge between the parasite and host cell membranes, referred to as the moving junction (MJ). The MJ is shaped by the assembly of AMA1 and RON2, as part of a complex involving additional RONs. While this essential process is well characterized in tachyzoites, the invasion process remains unexplored in bradyzoites, which form cysts and are responsible for chronic toxoplasmosis and contribute to the dissemination of the parasite between hosts. Here, we show that bradyzoites invade host cells in an MJ-dependent fashion but differ in protein composition from the tachyzoite MJ, relying instead on the paralogs AMA2 and AMA4. Functional characterization of AMA4 reveals its key role for cysts burden during the onset of chronic infection, while being dispensable for the acute phase. Immunizations with AMA1 and AMA4, alone or in complex with their rhoptry neck respective partners RON2 and RON2L1, showed that the AMA1–RON2 pair induces strong protection against acute and chronic infection, while the AMA4–RON2L1 complex targets more selectively the chronic form. Our study provides important insights into the molecular players of bradyzoite invasion and indicates that invasion of cyst-forming bradyzoites contributes to cyst burden. Furthermore, we validate AMA–RON complexes as potential vaccine candidates to protect against toxoplasmosis. [ABSTRACT FROM AUTHOR]

The etiological agent of syphilis, Treponema pallidum ssp. pallidum , is a highly invasive "stealth" pathogen that can evade the host immune response and persist within the host for decades. This obligate human pathogen is adept at establishing infection and surviving at sites within the host that have a multitude of competing microbes, sometimes including pathogens. One survival strategy employed by bacteria found at polymicrobial sites is elimination of competing microorganisms by production of antimicrobial peptides (AMPs). Antimicrobial peptides are low molecular weight proteins (miniproteins) that function directly via inhibition and killing of microbes and/or indirectly via modulation of the host immune response, which can facilitate immune evasion. In the current study, we used bioinformatics to show that approximately 7% of the T. pallidum proteome is comprised of miniproteins of 150 amino acids or less with unknown functions. To investigate the possibility that AMP production is an unrecognized defense strategy used by T. pallidum during infection, we developed a bioinformatics pipeline to analyze the complement of T. pallidum miniproteins of unknown function for the identification of potential AMPs. This analysis identified 45 T. pallidum AMP candidates; of these, Tp0451a and Tp0749 were subjected to further bioinformatic analyses to identify AMP critical core regions (AMPCCRs). Four potential AMPCCRs from the two predicted AMPs were identified and peptides corresponding to these AMPCCRs were experimentally confirmed to exhibit bacteriostatic and bactericidal activity against a panel of biologically relevant Gram-positive and Gram-negative bacteria. Immunomodulation assays performed under inflammatory conditions demonstrated that one of the AMPCCRs was also capable of differentially regulating expression of two pro-inflammatory chemokines [monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8)]. These findings demonstrate proof-of-concept for our developed AMP identification pipeline and are consistent with the novel concept that T. pallidum expresses AMPs to defend against competing microbes and modulate the host immune response. [ABSTRACT FROM AUTHOR]

The structure of the antigen‐binding fragment (Fab) of mouse monoclonal antibody 7H2.2 in complex with a 15‐residue fragment from the metalloproteinase sperm acrosomal SLLP1 binding protein (SAS1B), which is a molecular and cellular candidate for both cancer therapy and female contraception, has been determined at 2.75 Å resolution by single‐crystal X‐ray diffraction. Although the crystallization conditions contained the final 148 C‐terminal residues of SAS1B, the Fab was observed to crystallize in complex with a 15‐residue fragment corresponding to one of only two elements of secondary structure that are predicted to be ordered within the C‐terminal region of SAS1B. The antigen forms an amphipathic α‐helix that binds the 7H2.2 combining site via hydrophilic residues in an epitope that spans the length of the antigen α‐helix, with only two CH–π interactions observed along the edge of the interface between the antibody and antigen. Interestingly, the paratope contains two residues mutated away from the germline (YL32F and YH58R), as well as a ProH96‐ThrH97‐AspH98‐AspH99 insertion within heavy chain CDR3. The intact 7H2.2 antibody exhibits high affinity for the SAS1B antigen, with 1:1 binding and nanomolar affinity for both the SAS1B C‐terminal construct used for crystallization (3.38 ± 0.59 nM) and a 15‐amino‐acid synthetic peptide construct corresponding to the helical antigen observed within the crystal structure (1.60 ± 0.31 nM). The SAS1B–antibody structure provides the first structural insight into any portion of the subdomain architecture of the C‐terminal region of the novel cancer‐oocyte tumor surface neoantigen SAS1B and provides a basis for the targeted use of SAS1B. [ABSTRACT FROM AUTHOR]

Intedeukin-2 (IL-2) is an immunoregulatory cytokine that binds sequentially to the alpha (IL-2Rα), beta (IL-2Rβ), and common gamma chain ([γ.sub.c]) receptor subunits. Here we present the 2.8 angstrom crystal structure of a complex between human IL-2 and IL-2Rα, which interact in a docking mode distinct from that of other cytokine receptor complexes. IL-2Rα is composed of strandswapped 'sushi-like' domains, unlike the classical cytokine receptor fold. As a result of this domain swap, IL-2Rα uses a composite surface to dock into a groove on IL-2 that also serves as a binding site for antagonist drugs. With this complex, we now have representative structures for each class of hematopoietic cytokine receptor-docking modules., Interleukin-2 (IL-2), which is one of the first cytokines identified and a member of the four-helix bundle cytokine superfamily, acts at the heart of the immune response (1). IL-2 and [...]

Interleukin-6 (IL-6) is an immunoregulatory cytokine that activates a cell-surface signaling assembly composed of IL-6, the IL-6 α-receptor (IL-6Rα), and the shared signaling receptor gp130. The 3.65 angstrom-resolution structure of the extracellular signaling complex reveals a hexameric, interlocking assembly mediated by a total of 10 symmetry-related, thermodynamically coupled interfaces. Assembly of the hexameric complex occurs sequentially: IL-6 is first engaged by IL-6Rα and then presented to gp130 in the proper geometry to facilitate a cooperative transition into the high-affinity, signaling-competent hexamer. The quaternary structures of other IL-6/IL-12 family signaling complexes are likely constructed by means of a similar topological blueprint., Gp130 is a shared signal-transducing receptor for a family of more than 10 different four-helix-bundle cytokines, including IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin-M (OSM), and others [...]

Fatty acid–and retinol-binding proteins (FARs) belong to a unique family of excreted/secreted proteins (ESPs) found exclusively in nematodes. Much of our understanding of these proteins, however, is limited to their in vitro binding characteristics toward various fatty acids and retinol and has provided little insight into their in vivo functions or mechanisms. Recent research, however, has shown that FARs elicit an immunomodulatory role in plant and animal model systems, likely by sequestering lipids involved in immune signaling. This alludes to the intricate relationship between parasitic nematode effectors and their hosts. [ABSTRACT FROM AUTHOR]

The active site of copper-containing nitrite reductase (NiR), the carbonyl derivatives of the wild-type enzyme and the Asp98Asn variant are studied by Fourier transform infrared (FTIR) spectroscopy. NiR from Alcaligenes faecalis (AfNiR) is a trimeric multi-copper enzyme.Z

Parasitic nematodes cause significant morbidity and mortality globally. Excretory/secretory products (ESPs) such as fatty acid- and retinol- binding proteins (FARs) are hypothesized to suppress host immunity during nematode infection, yet little is known about their interactions with host tissues. Leveraging the insect parasitic nematode, Steinernema carpocapsae, we describe here the first in vivo study demonstrating that FARs modulate animal immunity, causing an increase in susceptibility to bacterial co-infection. Moreover, we show that FARs dampen key components of the fly immune response including the phenoloxidase cascade and antimicrobial peptide (AMP) production. Our data also reveal that FARs deplete lipid signaling precursors in vivo as well as bind to these fatty acids in vitro, suggesting that FARs elicit their immunomodulatory effects by altering the availability of lipid signaling molecules necessary for an efficient immune response. Collectively, these data support a complex role for FARs in immunosuppression in animals and provide detailed mechanistic insight into parasitism in phylum Nematoda. Author summary: A central aspect of parasitic nematode success is their ability to modify host biology, including evasion and/or subversion of host immunity. Modulation of host biology and the pathology caused by parasitic nematodes is largely effected through the release of proteins and small molecules. There are hundreds of proteins released by nematodes during an infection and few have been studied in detail. Fatty acid- and retinol-binding proteins (FARs) are a unique protein family, found only in nematodes and some bacteria, and are released during nematode infection. We report that nematode FARs from S. carpocapsae, C. elegans and A. ceylanicum dampen fly immunity decreasing resistance to infection. Mechanistically, this is achieved through modulation of the phenoloxidase cascade and antimicrobial peptide production. Furthermore, FARs alter the availability of lipid immune signaling precursors in vivo and show binding specificity in vitro. [ABSTRACT FROM AUTHOR]

The lipid kinase PI4KB, which generates phosphatidylinositol 4‐phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB‐associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogen–deuterium exchange mass spectrometry to characterize the c10orf76‐PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA‐dependent phosphorylation. Complex‐disrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76‐PI4KB complex is required for the replication of c10orf76‐dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76‐dependent increase in PI4P levels at the Golgi. Synopsis: Enteroviruses hijack host PI4KB and GBF1 to build replication organelles. This study reveals that the direct interaction between c10orf76 and PI4KB is required for c10orf76 recruitment to the Golgi and the replication of c10orf76‐dependent enteroviruses. c10orf76 is localized to the Golgi through a direct interaction with the kinase linker of PI4KB.c10orf76 regulates GBF1 and active Arf1 dynamics that control Golgi PI4P levels.Enteroviruses that rely on c10orf76 for replication depend on formation of the c10orf76‐PI4KB complex. [ABSTRACT FROM AUTHOR]

Several studies are conducted to explain the identification, as well as the characterization of the various binding sites on s100A7 (psoriasin), a member of the family of cancer signaling proteins. The protein is shown to play an extremely significant role as a therapeutic target for inflammation, as well as cancer.

Results indicate that exogenous ligands can bind to the oxidized type-1 site to restore the site's normal spectroscopic properties, while the reduced type-1 site does not bind exogenous ligands. Furthermore, the rates of electron transfer to the oxidized type-1 sites in the nitrite reductase variants are similar to native types, in the presence of exogenous ligands.

The phylogenetically divergent spirochete bacterium Treponema pallidum subsp. pallidum is the causative agent of syphilis. Central to the capacity of T. pallidum to establish infection is the ability of the pathogen to attach to a diversity of host cells. Many pathogenic bacteria employ leucine‐rich repeat (LRR) domain‐containing proteins to mediate protein–protein interactions, including attachment to host components and establishment of infection. Intriguingly, T. pallidum expresses only one putative LRR domain‐containing protein (Tp0225) with an unknown function. In an effort to ascribe a function to Tp0225, a comprehensive phylogenetic analysis was first performed; this investigation revealed that Tp0225 clusters with the pathogenic clade of treponemes. Its crystal structure was then determined to 2.0 Å resolution using Pt SAD phasing, which revealed a noncanonical architecture containing a hexameric LRR core with a discontinuous β‐sheet bridged by solvent molecules. Furthermore, a surface‐exposed, hydrophobic pocket, which was found in Tp0225 but is largely absent in canonical LRR domains from other pathogenic bacteria, may serve to coordinate a hydrophobic ligand. Overall, this study provides the first structural characterization of the sole LRR domain‐containing protein from T. pallidum and offers insight into the unique molecular landscape of this important human pathogen. [ABSTRACT FROM AUTHOR]

Summary: The Toxoplasma gondii locus mitochondrial association factor 1 (MAF1) encodes multiple paralogs, some of which mediate host mitochondrial association (HMA). Previous work showed that HMA was a trait that arose in T. gondii through neofunctionalization of an ancestral MAF1 ortholog. Structural analysis of HMA‐competent and incompetent MAF1 paralogs (MAF1b and MAF1a, respectively) revealed that both paralogs harbor an ADP ribose binding macro‐domain, with comparatively low (micromolar) affinity for ADP ribose. Replacing the 16 C‐terminal residues of MAF1b with those of MAF1a abrogated HMA, and we also show that only three residues in the C‐terminal helix are required for MAF1‐mediated HMA. Importantly these same three residues are also required for the in vivo growth advantage conferred by MAF1b, providing a definitive link between in vivo proliferation and manipulation of host mitochondria. Co‐immunoprecipitation assays reveal that the ability to interact with the mitochondrial MICOS complex is shared by HMA‐competent and incompetent MAF1 paralogs and mutants. The weak ADPr coordination and ability to interact with the MICOS complex shared between divergent paralogs may represent modular ancestral functions for this tandemly expanded and diversified T. gondii locus. [ABSTRACT FROM AUTHOR]

Treponema pallidum subspecies pallidum is the causative agent of syphilis, a chronic, multistage, systemic infection that remains a major global health concern. The molecular mechanisms underlying T. pallidum pathogenesis are incompletely understood, partially due to the phylogenetic divergence of T. pallidum. One aspect of T. pallidum that differentiates it from conventional Gram-negative bacteria, and is believed to play an important role in pathogenesis, is its unusual cell envelope ultrastructure; in particular, the T. pallidum peptidoglycan layer is chemically distinct, thinner and more distal to the outer membrane. Established functional roles for peptidoglycan include contributing to the structural integrity of the cell envelope and stabilization of the flagellar motor complex, which are typically mediated by the OmpA domain-containing family of proteins. To gain insight into the molecular mechanisms that govern peptidoglycan binding and cell envelope biogenesis in T. pallidum we report here the structural characterization of the putative OmpA-like domain-containing protein, Tp0624. Analysis of the 1.70 Å resolution Tp0624 crystal structure reveals a multi-modular architecture comprised of three distinct domains including a C-terminal divergent OmpA-like domain, which we show is unable to bind the conventional peptidoglycan component diaminopimelic acid, and a previously uncharacterized tandem domain unit. Intriguingly, bioinformatic analysis indicates that the three domains together are found in all orthologs from pathogenic treponemes, but are not observed together in genera outside Treponema. These findings provide the first structural insight into a multi-modular treponemal protein containing an OmpA-like domain and its potential role in peptidoglycan coordination and stabilization of the T. pallidum cell envelope. [ABSTRACT FROM AUTHOR]

Syphilis is a chronic disease caused by the bacterium Treponema pallidum subsp. pallidum. Treponema pallidum disseminates widely throughout the host and extravasates from the vasculature, a process that is at least partially dependent upon the ability of T. pallidum to interact with host extracellular matrix (ECM) components. Defining the molecular basis for the interaction between T. pallidum and the host is complicated by the intractability of T. pallidum to in vitro culturing and genetic manipulation. Correspondingly, few T. pallidum proteins have been identified that interact directly with host components. Of these, Tp0751 (also known as pallilysin) displays a propensity to interact with the ECM, although the underlying mechanism of these interactions remains unknown. Towards establishing the molecular mechanism of Tp0751-host ECM attachment, we first determined the crystal structure of Tp0751 to a resolution of 2.15 Å using selenomethionine phasing. Structural analysis revealed an eight-stranded beta-barrel with a profile of short conserved regions consistent with a non-canonical lipocalin fold. Using a library of native and scrambled peptides representing the full Tp0751 sequence, we next identified a subset of peptides that showed statistically significant and dose-dependent interactions with the ECM components fibrinogen, fibronectin, collagen I, and collagen IV. Intriguingly, each ECM-interacting peptide mapped to the lipocalin domain. To assess the potential of these ECM-coordinating peptides to inhibit adhesion of bacteria to host cells, we engineered an adherence-deficient strain of the spirochete Borrelia burgdorferi to heterologously express Tp0751. This engineered strain displayed Tp0751 on its surface and exhibited a Tp0751-dependent gain-of-function in adhering to human umbilical vein endothelial cells that was inhibited in the presence of one of the ECM-interacting peptides (p10). Overall, these data provide the first structural insight into the mechanisms of Tp0751-host interactions, which are dependent on the protein’s lipocalin fold. [ABSTRACT FROM AUTHOR]

Background: Identification of species-specific trypanosome molecules is important for laboratory- and field-based research into epidemiology and disease diagnosis. Although Trypanosoma congolense is the most important trypanosome pathogen of cattle in Africa, no species-specific molecules found in infective bloodstream forms (BSF) of the parasites have been identified, thus limiting development of diagnostic tests. Methods: Immuno-mass spectrometric methods were used to identify a protein that is recognized by a T. congolense-specific monoclonal antibody (mAb) Tc6/42.6.4. The identified molecule was expressed as a recombinant protein in E. coli and was tested in several immunoassays for its ability to interact with the mAb. The three dimensional structure of the protein was modeled and compared to crystal- and NMR-structures of the homologous proteins from T. cruzi and T. brucei respectively, in order to examine structural differences leading to the different immunoreactivity of the T. congolense molecule. Enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies produced by trypanosome-infected African cattle in order to assess the potential for use of T. congolense calflagin in a serodiagnostic assay. Results: The antigen recognized by the T. congolense-specific mAb Tc6/42.6.4 was identified as a flagellar calcium-binding protein, calflagin. The recombinant molecule showed immunoreactivity with the T. congolense-specific mAb confirming that it is the cognate antigen. Immunofluorescence experiments revealed that Ca2+ modulated the localization of the calflagin molecule in trypanosomes. Structural modelling and comparison with calflagin homologues from other trypanosomatids revealed four non-conserved regions on the surface of the T. congolense molecule that due to differences in surface chemistry and structural topography may form species-specific epitopes. ELISAs using the recombinant calflagin as antigen to detect antibodies in trypanosome-infected cattle showed that the majority of cattle had antibody responses. Area under the Receiver-Operating Characteristic (ROC) curves, associated with host IgG and IgM, were calculated to be 0.623 and 0.709 respectively, indicating a positive correlation between trypanosome infection and the presence of anti-calflagin antibodies. Conclusions: While calflagin is conserved among different species of African trypanosomes, our results show that T. congolense calflagin possesses unique epitopes that differentiate this protein from homologues in other trypanosome species. MAb Tc6/42.6.4 has clear utility as a laboratory tool for identifying T. congolense. T. congolense calflagin has potential as a serodiagnostic antigen and should be explored further for its utility in antigen-detection assays for diagnosis of cattle infections. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum is an obligate intracellular protozoan parasite that employs a highly sophisticated mechanism to access the protective environment of the host cells. Key to this mechanism is the formation of an electron dense ring at the parasite-host cell interface called the Moving Junction (MJ) through which the parasite invades. The MJ incorporates two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, the latter one being targeted to the host cell membrane during invasion. Crystal structures of AMA1 have shown that a partially mobile loop, termed the DII loop, forms part of a deep groove in domain I and overlaps with the RON2 binding site. To investigate the mechanism by which the DII loop influences RON2 binding, we measured the kinetics of association and dissociation and binding equilibria of a PfRON2sp1 peptide with both PfAMA1 and an engineered form of PfAMA1 where the flexible region of the DII loop was replaced by a short Gly-Ser linker (ΔDII-PfAMA1). The reactions were tracked by fluorescence anisotropy as a function of temperature and concentration and globally fitted to acquire the rate constants and corresponding thermodynamic profiles. Our results indicate that both PfAMA1 constructs bound to the PfRON2sp1 peptide with the formation of one intermediate in a sequential reversible reaction: A↔B↔C. Consistent with Isothermal Titration Calorimetry measurements, final complex formation was enthalpically driven and slightly entropically unfavorable. Importantly, our experimental data shows that the DII loop lengthened the complex half-life time by 18-fold (900 s and 48 s at 25°C for Pf and ΔDII-Pf complex, respectively). The longer half-life of the Pf complex appeared to be driven by a slower dissociation process. These data highlight a new influential role for the DII loop in kinetically locking the functional binary complex to enable host cell invasion. [ABSTRACT FROM AUTHOR]