Acquired protection from
P
lasmodium falciparum
malaria takes years to develop, probably reflecting the ability of the parasites to evade immunity. A recent example of this is the binding of the F
c
...region of
IgM
to
VAR2CSA
‐type
PfEMP1
. This interferes with specific
IgG
recognition and phagocytosis of opsonized infected erythrocytes (
IEs
) without compromising the placental
IE
adhesion mediated by this
PfEMP1
type.
IgM
also binds via F
c
to several other
PfEMP1
proteins, where it has been proposed to facilitate rosetting (binding of uninfected erythrocytes to a central
IE
). To further dissect the functional role of
F
c
‐mediated
IgM
binding to
PfEMP1
, we studied the
PfEMP1
protein
HB3VAR06
, which mediates rosetting and binds
IgM
. Binding of
IgM
to this
PfEMP1
involved the
F
c
domains
C
μ3‐
C
μ4 in
IgM
and the penultimate
DBL
domain (
DBLζ2
) at the
C
‐terminus of
HB3VAR06
. However,
IgM
binding did not inhibit specific
IgG
labelling of
HB3VAR06
or shield
IgG
‐opsonized IEs from phagocytosis. Instead,
IgM
was required for rosetting, and each pentameric IgM molecule could bind two
HB3VAR
06 molecules. Together, our data indicate that the primary function of
F
c
‐mediated
IgM
binding in rosetting is not to shield
IE
from specific
IgG
recognition and phagocytosis as in
VAR2CSA
‐type
PfEMP1
. Rather, the function appears to be strengthening of
IE
–erythrocyte interactions. In conclusion, our study provides new evidence on the molecular details and functional significance of rosetting, a long‐recognized marker of parasites that cause severe
P
. falciparum
malaria.
•Development of a secretory HEK293 expression system for the production of all human IGFBPs.•Generation of mature full-length, PTM containing IGFBPs.•Proteins display little or no degradation ...products.•High-affinity interactions to IGFs are observed.•A method to obtain human IGFBPs for subsequent investigations of the IGFBP interaction matrix.
Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2.
The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached ...kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.
S100B is a member of the S100 subfamily of EF-hand proteins that has been implicated in malignant melanoma and neurodegenerative conditions such as Alzheimer's and Parkinson's disease. ...Calcium-induced conformational changes expose a hydrophobic binding cleft, facilitating interactions with a wide variety of nuclear, cytoplasmic, and extracellular target proteins. Previously, peptides derived from CapZ, p53, NDR, HDM2 and HDM4 have been shown to interact with S100B in a calcium-dependent manner. However, the thermodynamic and kinetic basis of these interactions remains largely unknown. To gain further insight, these peptides were screened against the S100B protein using isothermal titration calorimetry and nuclear magnetic resonance. All peptides were found to have binding affinities in the low micromolar to nanomolar range. Binding-induced changes in the line shapes of S100B backbone
1
H and
15
N were monitored to obtain the dissociation constants and the kinetic binding parameters. The large microscopic K
on
rate constants observed in this study, K
on
≥1×10
7
M
-1
s
-1
, suggest that S100B utilizes a “fly casting mechanism” in the recognition of these peptide targets.
Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many ...of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.
Background: We searched for novel extracellular proteins in Streptococcus pyogenes.
Results: A protein (sHIP) protecting S. pyogenes against antibacterial activity was identified. Its structure was determined, and severe S. pyogenes infections were connected with elevated antibody titers against this protein.
Conclusion: We identified a protein with unique structure and impact on S. pyogenes virulence.
Significance: This work increases our understanding of S. pyogenes pathogenesis.
Chromosome integrity depends on DNA structure‐specific processing complexes that resolve DNA entanglement between sister chromatids. If left unresolved, these entanglements can generate either ...chromatin bridging or ultrafine DNA bridging in the anaphase of mitosis. These bridge structures are defined by the presence of the PICH protein, which interacts with the BEND3 protein in mitosis. To obtain structural insights into PICH–BEND3 complex formation at the atomic level, their respective NTPR and BD1 domains were cloned, overexpressed and crystallized using 1.56 M ammonium sulfate as a precipitant at pH 7.0. The protein complex readily formed large hexagonal crystals belonging to space group P6122, with unit‐cell parameters a = b = 47.28, c = 431.58 Å and with one heterodimer in the asymmetric unit. A complete multiwavelength anomalous dispersion (MAD) data set extending to 2.2 Å resolution was collected from a selenomethionine‐labelled crystal at the Swiss Light Source.
The interaction between PICH and BEND3 is dissected and the domains involved in their interaction have been crystallized.
Streptococcus pyogenes is one of the most significant bacterial pathogens in the human population mostly causing superficial and uncomplicated infections (pharyngitis and impetigo) but also invasive ...and life-threatening disease. We have previously identified a virulence determinant, protein sHIP, which is secreted at higher levels by an invasive compared to a non-invasive strain of S. pyogenes. The present work presents a further characterization of the structural and functional properties of this bacterial protein. Biophysical and structural studies have shown that protein sHIP forms stable tetramers both in the crystal and in solution. The tetramers are composed of four helix-loop-helix motifs with the loop regions connecting the helices displaying a high degree of flexibility. Owing to interactions at the tetramer interface, the observed tetramer can be described as a dimer of dimers. We identified three residues at the tetramer interface (Leu84, Leu88, Tyr95), which due to largely non-polar side-chains, could be important determinants for protein oligomerization. Based on these observations, we produced a sHIP variant in which these residues were mutated to alanines. Biophysical experiments clearly indicated that the sHIP mutant appear only as dimers in solution confirming the importance of the interfacial residues for protein oligomerisation. Furthermore, we could show that the sHIP mutant interacts with intact histidine-rich glycoprotein (HRG) and the histidine-rich repeats in HRG, and inhibits their antibacterial activity to the same or even higher extent as compared to the wild type protein sHIP. We determined the crystal structure of the sHIP mutant, which, as a result of the high quality of the data, allowed us to improve the existing structural model of the protein. Finally, by employing NMR spectroscopy in solution, we generated a model for the complex between the sHIP mutant and an HRG-derived heparin-binding peptide, providing further molecular details into the interactions involving protein sHIP.
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