Insights into the role of metal ions

Die vorliegende Arbeit untersucht die Frage: Welche Wirkung haben Metall-Ionen auf die Struktur, Dynamik und Wechselwirkungen des Proteins ShhN, der N-terminalen Signal- Domäne von Sonic Hedgehog? Zur Beantwortung wurden molekulare Strukturen von ShhN mit verschiedenen Computer-gestützten Methoden analysiert. Die Analysen sagen einige überraschende Eigenschaften von ShhN vorher. Zuerst legen die Analysen nahe, dass ShhN eine Zink-Protease ist, deren katalytisches Zentrum durch die Bindung von Calcium-Ionen reguliert wird. Speziell ändert die Bindung des zweiten Calcium-Ions die Konformation des Zink-Zentrums und beeinflusst so vermut- lich die Bindung des Substrats. Diese Konformationsänderung kaskadiert vom Calcium- Ion zum katalytisch Zink-Zentrum über mehrere Residuen und destabilisiert die Substrat- Bindestelle und das Zink-gebundene katalytische Wasser-Molekül. Änderungen des elek- trostatischen Potenzials um das Zink-Zentrum in Abhängigkeit von der Calcium-Bindung weisen hin auf eher unpolare Substrate, z.B. den C-Terminus von ShhN. ShhN könnte also ShhN-Moleküle abbauen, und auf diese Weise seinen Konzentrationsgradienten än- dern. Die Implikationen des beschriebenen neuartigen Mechanismus der Schaltung von ShhN über Calcium-Ionen für die biologische Funktion des Proteins sind noch weitgehend unverstanden, und erfordern weitere Forschung. Die Flexibilität von ShhN-Monomeren und -Dimeren hängt stark ab von der Zahl der gebun- denen Calcium-Ionen. Im Besonderen sind die Calcium-bindenden Schleifen von ShhN davon betroffen. ShhN-Dimere werden stabilisiert durch das Cardin-Weintraub-Motiv am N-Terminus und durch Wechselwirkungen mit hydrophoben Residuen an der Schnittstelle zwischen den Monomeren. Die schwächere Konservierung von I48 in Wirbeltieren kön- nte ein Hinweis sein auf eine wichtige Rolle dieses Residuums in der Oligomerisierung von ShhN. Die Nähe von Calcium-Ionen zur Dimerisierungs-Region sorgt für ein positives elektrostatisches Potenzial und könnte so die Bindung von Proteoglykanen fördern. Ins- gesamt wird ein Modell der Multimerisierung von ShhN vorgeschlagen, das über mehrere Kontrollmechanismen verfügt, die experimentell getestet werden sollten.The present work studies the effect of metal ions on the structure, dynamics and inter- actions of the protein ShhN, the N-terminal signaling domain of Sonic Hedgehog. To accomplish this task, molecular structures of ShhN proteins were analyzed with a set of computational methods, revealing new features of ShhN proteins. The results suggest that, ShhN is an enzyme with a zinc catalytic center that is regulated by the binding of the calcium ions. Explicitly, the binding of the second calcium ion involves a conformational change that is accompanied by a significant perturbation of the putative catalytic center, possibly affecting substrate stabilization. The dragging of E127 towards the calcium center implies the pulling of H135 with it and the disruption of the hydrogen bond between G128 and H141. Besides, the distance between residues E177 and H135 increases and therefore, the well-defined position of the catalytic water molecule is lost destabilizing the zinc environment. Electrostatic potential differences among calcium states suggest the possible binding of nonpolar substrates. One of the predictions is that ShhN autodegradates tuning its own concentration gradient. This possibility does not rule out, of course, the existence of other mechanisms that govern ShhN concentration gradient. The novel switching mechanism proposed could have many implications in the biological function of HhN proteins, but these are not well understood and require further research. Both ShhN monomers and dimers show a flexibility pattern that strongly depends on the number of calcium ions. Specially, calcium binding loops reflect this behavior. The Cardin- Weintraub motif located within the N-terminal of ShhN proteins together with buried hydrophobic residues at the interface lead to an stable complex that enhances ShhN dimerization. The lower degree of conservation of I48 in vertebrate homologs might indicate that this is a hot spot residue with an important role in ShhN oligomerization. The presence of the calcium ions at the dimeric interface can promote ShhN-proteoglycan interactions providing a large positively charged region which is the ideal scenario for the binding of these molecules. Taken all together, a multimerization model where different levels of interaction can control the way that ShhN multimers form is proposed. However, this model has yet to be tested

Stromal cells support the survival of human primary chronic lymphocytic leukemia (CLL) cells through Lyn-driven extracellular vesicles

Introduction In chronic lymphocytic leukemia (CLL), the tumor cells receive survival support from stromal cells through direct cell contact, soluble factors and extracellular vesicles (EVs). The protein tyrosine kinase Lyn is aberrantly expressed in the malignant and stromal cells in CLL tissue. We studied the role of Lyn in the EV-based communication and tumor support. Methods We compared the Lyn-dependent EV release, uptake and functionality using Lyn-proficient (wild-type) and -deficient stromal cells and primary CLL cells. Results Lyn-proficient cells caused a significantly higher EV release and EV uptake as compared to Lyn-deficient cells and also conferred stronger support of primary CLL cells. Proteomic comparison of the EVs from Lyn-proficient and -deficient stromal cells revealed 70 significantly differentially expressed proteins. Gene ontology studies categorized many of which to organization of the extracellular matrix, such as collagen, fibronectin, fibrillin, Lysyl oxidase like 2, integrins and endosialin (CD248). In terms of function, a knockdown of CD248 in Lyn+ HS-5 cells resulted in a diminished B-CLL cell feeding capacity compared to wildtype or scrambled control cells. CD248 is a marker of certain tumors and cancer-associated fibroblast (CAF) and crosslinks fibronectin and collagen in a membrane-associated context. Conclusion Our data provide preclinical evidence that the tyrosine kinase Lyn crucially influences the EV-based communication between stromal and primary B-CLL cells by raising EV release and altering the concentration of functional molecules of the extracellular matrix

HPIPred: Host-pathogen interactome prediction with phenotypic scoring

Protein-protein interactions (PPIs) are involved in most cellular processes. Unfortunately, current knowl-edge of host-pathogen interactomes is still very limited. Experimental methods used to detect PPIs have several limitations, including increasing complexity and economic cost in large-scale screenings. Hence, computational methods are commonly used to support experimental data, although they generally suffer from high false-positive rates. To address this issue, we have created HPIPred, a host-pathogen PPI pre-diction tool based on numerical encoding of physicochemical properties. Unlike other available methods, HPIPred integrates phenotypic data to prioritize biologically meaningful results. We used HPIPred to screen the entire Homo sapiens and Pseudomonas aeruginosa PAO1 proteomes to generate a host -pathogen interactome with 763 interactions displaying a highly connected network topology. Our predic-tive model can be used to prioritize protein-protein interactions as potential targets for antibacterial drug development. Available at: https://github.com/SysBioUAB/hpi_predictor.(c) 2022 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY-NC-ND license (http://creative-commons.org/licenses/by-nc-nd/4.0/)

How Do Molecular Tweezers Bind to Proteins? Lessons from X-ray Crystallography

To understand the biological relevance and mode of action of artificial protein ligands, crystal structures with their protein targets are essential. Here, we describe and investigate all known crystal structures that contain a so-called “molecular tweezer” or one of its derivatives with an attached natural ligand on the respective target protein. The aromatic ring system of these compounds is able to include lysine and arginine side chains, supported by one or two phosphate groups that are attached to the half-moon-shaped molecule. Due to their marked preference for basic amino acids and the fully reversible binding mode, molecular tweezers are able to counteract pathologic protein aggregation and are currently being developed as disease-modifying therapies against neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. We analyzed the corresponding crystal structures with 14-3-3 proteins in complex with mono- and diphosphate tweezers. Furthermore, we solved crystal structures of two different tweezer variants in complex with the enzyme Δ1-Pyrroline-5-carboxyl-dehydrogenase (P5CDH) and found that the tweezers are bound to a lysine and methionine side chain, respectively. The different binding modes and their implications for affinity and specificity are discussed, as well as the general problems in crystallizing protein complexes with artificial ligands

Effect of binding on ShhN structure.

<p>Structures taken from MD trajectories for Ca2 (grey; based on PDB entry 3d1m with two ) and Ca0 (red; 1vhh). Shown are sampled structures close to the cluster centers of Ca2 and Ca0 ensembles. (A) Overview and comparison of full ShhN structures in Ca2 and Ca0 states. Position of calcium ions marked by green mesh. Calcium binding pocket formed by loops , breaks up as is removed (transition grey to red). Neighboring loop is also affected although it does not co-ordinate . (B) Close-up of (green spheres) binding site in Ca2 state. Calcium ions are surrounded by a cage of anionic side chains. (C) Same region as in panel B, but now in Ca0 state. Note the large distance differences of anionic groups between B and C.</p

Phylogenetic tree of all 30 reviewed full length Hedgehog proteins from UniProtKB.

<p>The vertebrate Hedgehogs (bottom subtree) are clearly separated from the <i>Drosophila</i> Hedgehogs (top subtree). In all vertebrates the full catalytic motif is absolutely conserved (red), except in rat with one conservative exchange (blue).</p

Signaling Domain of Sonic Hedgehog as Cannibalistic Calcium-Regulated Zinc-Peptidase

<div><p>Sonic Hedgehog (Shh) is a representative of the evolutionary closely related class of Hedgehog proteins that have essential signaling functions in animal development. The N-terminal domain (ShhN) is also assigned to the group of LAS proteins (LAS = Lysostaphin type enzymes, D-Ala-D-Ala metalloproteases, Sonic Hedgehog), of which all members harbor a structurally well-defined center; however, it is remarkable that ShhN so far is the only LAS member without proven peptidase activity. Another unique feature of ShhN in the LAS group is a double- center close to the zinc. We have studied the effect of these calcium ions on ShhN structure, dynamics, and interactions. We find that the presence of calcium has a marked impact on ShhN properties, with the two calcium ions having different effects. The more strongly bound calcium ion significantly stabilizes the overall structure. Surprisingly, the binding of the second calcium ion switches the putative catalytic center from a state similar to LAS enzymes to a state that probably is catalytically inactive. We describe in detail the mechanics of the switch, including the effect on substrate co-ordinating residues and on the putative catalytic water molecule. The properties of the putative substrate binding site suggest that ShhN could degrade other ShhN molecules, e.g. by cleavage at highly conserved glycines in ShhN. To test experimentally the stability of ShhN against autodegradation, we compare two ShhN mutants <i>in vitro</i>: (1) a ShhN mutant unable to bind calcium but with putative catalytic center intact, and thus, according to our hypothesis, a constitutively active peptidase, and (2) a mutant carrying additionally mutation E177A, i.e., with the putative catalytically active residue knocked out. The <i>in vitro</i> results are consistent with ShhN being a cannibalistic zinc-peptidase. These experiments also reveal that the peptidase activity depends on .</p></div

<i>In vitro</i> tests of ShhN mutant stabilities against proteolysis.

<p>The logarithm of protein content (relative to maximum protein content) is plotted over time. Proteins are (E177A, red) and (E177, blue). Straight red and blue lines are least squares fits to the measurements, shaded areas around these lines are 95% confidence intervals for the corresponding linear models. All data refer to measurements at .</p

Switch mechanism triggered by Ca2 calcium ion.

<p>(A) ShhN zinc center in states Ca0 (X-ray structure 1vhh, red), Ca1 (X-ray structure 3n1r, blue), Ca2 (X-ray structure 3d1m, black). Putative catalytic water from 1vhh is close to the zinc ion. From Ca0 to Ca1 and Ca2, E127 carboxylate is drawn towards and drags H-bound H135 side chain with it, away from substrate and the active E177. While Ca0 and Ca1 superimpose well, Ca2 is clearly different. (B) Central components of the switch mechanisms in states Ca0 and Ca2. (C) Distances between H-bonded E127 carboxylate-O and H135 imidazole-proton, and between substrate-clamping side chains of H135 and catalytically active E177. Red (Ca0) and black (Ca2) points are sampled by MD simulations. Green triangle (Ca0) and green square (Ca2) are the corresponding values directly taken from X-ray structures 1vhh and 3d1m, respectively.</p

Zinc centers of peptidases and ShhN.

<p>The four examples shown are Thermolysin (A), ShhN (B), and LAS peptidases <i>Streptomyces albus G</i> D-Ala-D-Ala Carboxypeptidase (PDB: 1lbu) (C), and L-alanoyl-D-glutamate endopeptidase of a bacteriophage (PDB: 2vo9) (D). Numbers on dashed lines are characteristic distances in 0.1 nm.</p