Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands

: Thrombin is the key enzyme of the entire hemostatic process since it is able to exert both procoagulant and anticoagulant functions; therefore, it represents an attractive target for the developments of biomolecules with therapeutic potential. Thrombin can perform its many functional activities because of its ability to recognize a wide variety of substrates, inhibitors, and cofactors. These molecules frequently are bound to positively charged regions on the surface of protein called exosites. In this review, we carried out extensive analyses of the structural determinants of thrombin partnerships by surveying literature data as well as the structural content of the Protein Data Bank (PDB). In particular, we used the information collected on functional, natural, and synthetic molecular ligands to define the anatomy of the exosites and to quantify the interface area between thrombin and exosite ligands. In this framework, we reviewed in detail the specificity of thrombin binding to aptamers, a class of compounds with intriguing pharmaceutical properties. Although these compounds anchor to protein using conservative patterns on its surface, the present analysis highlights some interesting peculiarities. Moreover, the impact of thrombin binding aptamers in the elucidation of the cross-talk between the two distant exosites is illustrated. Collectively, the data and the work here reviewed may provide insights into the design of novel thrombin inhibitors

Structural and functional analysis of the simultaneous binding of two duplex/quadruplex aptamers to human α-thrombin

: The long-range communication between the two exosites of human α-thrombin (thrombin) tightly modulates the protein-effector interactions. Duplex/quadruplex aptamers represent an emerging class of very effective binders of thrombin. Among them, NU172 and HD22 aptamers are at the forefront of exosite I and II recognition, respectively. The present study investigates the simultaneous binding of these two aptamers by combining a structural and dynamics approach. The crystal structure of the ternary complex formed by the thrombin with NU172 and HD22_27mer provides a detailed view of the simultaneous binding of these aptamers to the protein, inspiring the design of novel bivalent thrombin inhibitors. The crystal structure represents the starting model for molecular dynamics studies, which point out the cooperation between the binding at the two exosites. In particular, the binding of an aptamer to its exosite reduces the intrinsic flexibility of the other exosite, that preferentially assumes conformations similar to those observed in the bound state, suggesting a predisposition to interact with the other aptamer. This behaviour is reflected in a significant increase of the anticoagulant activity of NU172 when the inactive HD22_27mer is bound to exosite II, providing a clear evidence of the synergic action of the two aptamers

The characterization of Thermotoga maritima Arginine Binding Protein variants demonstrates that minimal local strains have an important impact on protein stability

The Ramachandran plot is a versatile and valuable tool that provides fundamental information for protein structure determination, prediction, and validation. The structural/thermodynamic effects produced by forcing a residue to adopt a conformation predicted to be forbidden were here explored using Thermotoga maritima Arginine Binding Protein (TmArgBP) as model. Specifically, we mutated TmArgBP Gly52 that assumes a conformation believed to be strictly disallowed for non-Gly residues. Surprisingly, the crystallographic characterization of Gly52Ala TmArgBP indicates that the structural context forces the residue to adopt a non-canonical conformation never observed in any of the high-medium resolution PDB structures. Interestingly, the inspection of this high resolution structure demonstrates that only minor alterations occur. Nevertheless, experiments indicate that Gly52 replacements in TmArgBP produce destabilizations comparable to those observed upon protein truncation or dissection in domains. Notably, we show that force-fields commonly used in computational biology do not reproduce this non-canonical state. Using TmArgBP as model system we here demonstrate that the structural context may force residues to adopt conformations believed to be strictly forbidden and that barely detectable alterations produce major destabilizations. Present findings highlight the role of subtle strains in governing protein stability. A full understanding of these phenomena is essential for an exhaustive comprehension of the factors regulating protein structures

Molecular dynamics simulations shed light on the cooperative mechanisms generated by the simultaneous binding of aptamers at the two exosites of thrombin”

Molecular dynamics simulations shed light on the cooperative mechanisms generated by the simultaneous binding of aptamers at the two exosites of thrombin Human α-thrombin is a trypsin-like serine protease endowed with the unique ability to convert soluble fibrinogen in insoluble fibrin clot. In addition to the active site, this enzyme owns two electropositive regions, exosite I and II, located at opposite sides of its globular shape [1]. The narrow substrate specificity of thrombin and its ability to change function are regulated by the exosite binding to different cofactors and modulators [2]. A special class of thrombin exosite synthetic ligands is represented by G-quadruplex anticoagulant aptamers, which are short single stranded DNA or RNA oligonucleotides that bind their targets with very high affinity and specificity [3]. The minimal 15mer DNA aptamer, named TBA, is the first and the most studied anti-thrombin aptamer. Based on several X-ray studies, this aptamer was established to bind the fibrinogen-binding site of thrombin (exosite I) by a pincer-like recognition mechanism involving the two TT loops [4]. The addition of a duplex motif to the G-quadruplex module has produced a new generation of aptamers with higher affinity against thrombin compared to TBA [5]. Among them, an aptamer, named HD22_27mer, recognizes exosite II with both quadruplex and duplex domains [6].In the last years, great attention has been paid to the study of the effects of the simultaneous binding of two ligands on the two exosites of thrombin. Biochemical studies have suggested that thrombin is an allosterically modulated enzyme: an interplay between its two exosites as well as between the exosites and the active site has been highlighted [7]. Furthermore, the crystallographic structures of two thrombin ternary complexes, in which exosite II is bound to HD22_27mer and exosite I interacts with TBA-like aptamers, were solved and gave structural information on the effects of the simultaneous binding of two aptamers to thrombin exosites [8]. Here we present the results of an extensive molecular dynamics study performed on free thrombin and on its binary and ternary complexes with TBA and HD22_27mer in the absence of the PPACK inhibitor that is covalently bound to the protein active site in all the crystallographic α-thrombin models. This study revealed that, in the absence of any influence of the crystal packing, an inter-exosite cross-talk and an active site-exosite communication may occur in these systems. Details will be discussed at the Meeting.[1] E. Di Cera, J Thromb Haemost., 5, (2007), 196-202.[2] S. Krishnaswamy, J Thromb Haemost., 3, (2005), 54-67.[3] A.D. Keefe, S. Pai, A. Ellington, Nat Rev Drug Discov., 9, (2010), 537-550.[4] I. Russo Krauss, A. Merlino, A. Randazzo, E. Novellino, L. Mazzarella, F. Sica, Nucleic Acids Res., 40, (2012), 8119-8128.[5] I. Russo Krauss, V. Napolitano, L. Petraccone, R. Troisi, V. Spiridonova, C.A. Mattia, F. Sica, Int J Biol Macromol., 107, (2018), 1697-1705.[6] I. Russo Krauss, A. Pica, A. Merlino, L. Mazzarella, F. Sica, Acta Cryst. D, 69, (2013), 2403-2411.[7] N.S. Petrera, A.R. Stafford, B.A. Leslie, C.A. Kretz, J.C. Fredenburgh, J.I. Weitz, J Biol Chem., 284, (2009), 25620-25629.[8] A. Pica, I. Russo Krauss, V. Parente, H. Tateishi-Karimata, S. Nagatoishi, K. Tsumoto, N. Sugimoto, F. Sica, Nucleic Acids Res., 45, (2017), 461-46

Alphafold Predictions Provide Insights into the Structural Features of the Functional Oligomers of All Members of the KCTD Family

Oligomerization endows proteins with some key properties such as extra-stabilization, long-range allosteric regulation(s), and partnerships not accessible to their monomeric counterparts. How oligomerization is achieved and preserved during evolution is a subject of remarkable scientific relevance. By exploiting the abilities of the machine-learning algorithms implemented in AlphaFold (AF) in predicting protein structures, herein, we report a comprehensive analysis of the structural states of functional oligomers of all members of the KCTD protein family. Interestingly, our approach led to the identification of reliable three-dimensional models for the pentameric states of KCNRG, KCTD6, KCTD4, KCTD7, KCTD9, and KCTD14 and possibly for KCTD11 and KCTD21 that are involved in key biological processes and that were previously uncharacterized from a structural point of view. Although for most of these proteins, the CTD domains lack any sequence similarity, they share some important structural features, such as a propeller-like structure with a central cavity delimited by five exposed and regular β-strands. Moreover, the structure of the related proteins KCTD7 and KCTD14, although pentameric, appears to be characterized by a different organization of the CTD region, with the five chains forming a circle-like structure with a large cavity. Our predictions also suggest that other members of the family, such as KCTD10, KCTD13, and TNFAIP1, present a strong propensity to assume dimeric states. Although the structures of the functional oligomers reported herein represent models that require additional validations, they provide a consistent and global view of KCTD protein oligomerization

The Structural Versatility of the BTB Domains of KCTD Proteins and Their Recognition of the GABAB Receptor

Several recent investigations have demonstrated that members of the KCTD (Potassium Channel Tetramerization Domain) protein family are involved in fundamental processes. However, the paucity of structural data available on these proteins has frequently prevented the definition of their biochemical role(s). Fortunately, this scenario is rapidly changing as, in very recent years, several crystallographic structures have been reported. Although these investigations have provided very important insights into the function of KCTDs, they have also raised some puzzling issues. One is related to the observation that the BTB (broad-complex, tramtrack, and bric-à-brac) domain of these proteins presents a remarkable structural versatility, being able to adopt a variety of oligomeric states. To gain insights into this intriguing aspect, we performed extensive molecular dynamics simulations on several BTB domains of KCTD proteins in different oligomeric states (monomers, dimers, tetramers, and open/close pentamers). These studies indicate that KCTD-BTB domains are stable in the simulation timescales, even in their monomeric forms. Moreover, simulations also show that the dynamic behavior of open pentameric states is strictly related to their functional roles and that different KCTDs may form stable hetero-oligomers. Molecular dynamics (MD) simulations also provided a dynamic view of the complex formed by KCTD16 and the GABAB2 receptor, whose structure has been recently reported. Finally, simulations carried out on the isolated fragment of the GABAB2 receptor that binds KCTD16 indicate that it is able to assume the local conformation required for the binding to KCTD

Development of a Protein Scaffold for Arginine Sensing Generated through the Dissection of the Arginine-Binding Protein from Thermotoga maritima

Arginine is one of the most important nutrients of living organisms as it plays a major role in important biological pathways. However, the accumulation of arginine as consequence of metabolic defects causes hyperargininemia, an autosomal recessive disorder. Therefore, the efficient detection of the arginine is a field of relevant biomedical/biotechnological interest. Here, we developed protein variants suitable for arginine sensing by mutating and dissecting the multimeric and multidomain structure of Thermotoga maritima arginine-binding protein (TmArgBP). Indeed, previous studies have shown that TmArgBP domain-swapped structure can be manipulated to generate simplified monomeric and single domain scaffolds. On both these stable scaffolds, to measure tryptophan fluorescence variations associated with the arginine binding, a Phe residue of the ligand binding pocket was mutated to Trp. Upon arginine binding, both mutants displayed a clear variation of the Trp fluorescence. Notably, the single domain scaffold variant exhibited a good affinity (~3 µM) for the ligand. Moreover, the arginine binding to this variant could be easily reverted under very mild conditions. Atomic-level data on the recognition process between the scaffold and the arginine were obtained through the determination of the crystal structure of the adduct. Collectively, present data indicate that TmArgBP scaffolds represent promising candidates for developing arginine biosensors

The temporal correlation between positive testing and death in Italy: From the first phase to the later evolution of the COVID-19 pandemic

BACKGROUND AND AIM: After the global spread of the coronavirus disease 2019 (COVID-19), research has concentrated its efforts on several aspects of the epidemiological burden of pandemic. In this frame, the presented study follows a previous analysis of the temporal link between cases and deaths during the first epidemic wave (Phase 1) in Italy (March-June 2020). METHODS: We here analyze the COVID-19 epidemic in the time span from March 2020 to June 2021. RESULTS: The elaboration of the curves of cases and deaths allows identifying the temporal shift between the positive testing and the fatal event, which corresponds to one week from W2 to W33, two weeks from W34 to W41, and three weeks from W42 to W67. Based on this finding, we calculate the Weekly Lethality Rate (WLR). The WLR was grossly overestimated (~13.5%) in Phase 1, while a mean value of 2.6% was observed in most of Phase 2 (starting from October 2020), with a drop to 1.4% in the last investigated weeks. CONCLUSIONS: Overall, these findings offer an interesting insight into the magnitude and time evolution of the lethality burden attributable to COVID-19 during the entire pandemic period in Italy. In particular, the analysis highlighted the impact of the effectiveness of public health and social measures, of changes in disease management, and of preventive strategies over time. (www.actabiomedica.it

A Structure-Based Mechanism for the Denaturing Action of Urea, Guanidinium Ion and Thiocyanate Ion

An exhaustive analysis of all the protein structures deposited in the Protein Data Bank, here performed, has allowed the identification of hundredths of protein-bound urea molecules and the structural characterization of such binding sites. It emerged that, even though urea molecules are largely involved in hydrogen bonds with both backbone and side chains, they are also able to make van der Waals contacts with nonpolar moieties. As similar findings have also been previously reported for guanidinium and thiocyanate, this observation suggests that promiscuity is a general property of protein denaturants. Present data provide strong support for a mechanism based on the protein-denaturant direct interactions with a denaturant binding model to equal and independent sites. In this general framework, our investigations also highlight some interesting insights into the different denaturing power of urea compared to guanidinium/thiocyanate

KCTD5 is endowed with large, functionally relevant, interdomain motions

<p>The KCTD family is an emerging class of proteins that are involved in important biological processes whose biochemical and structural properties are rather poorly characterized or even completely undefined. We here used KCTD5, the only member of the family with a known three-dimensional structure, to gain insights into the intrinsic structural stability of the C-terminal domain (CTD) and into the mutual dynamic interplay between the two domains of the protein. Molecular dynamics (MD) simulations indicate that in the simulation timescale (120 ns), the pentameric assembly of the CTD is endowed with a significant intrinsic stability. Moreover, MD analyses also led to the identification of exposed β-strand residues. Being these regions intrinsically sticky, they could be involved in the substrate recognition. More importantly, simulations conducted on the full-length protein provide interesting information of the relative motions between the BTB domain and the CTD of the protein. Indeed, the dissection of the overall motion of the protein is indicative of a large interdomain twisting associated with limited bending movements. Notably, MD data indicate that the entire interdomain motion is pivoted by a single residue (Ser150) of the hinge region that connects the domains. The functional relevance of these motions was evaluated in the context of the functional macromolecular machinery in which KCTD5 is involved. This analysis indicates that the interdomain twisting motion here characterized may be important for the correct positioning of the substrate to be ubiquitinated with respect to the other factors of the ubiquitination machinery.</p