Tyrosine-O-sulfation is a widespread affinity enhancer among thrombin interactors.

Tyrosine-O-sulfation is a common post-translational modification (PTM) of proteins following the cellular secretory pathway. First described in human fibrinogen, tyrosine-O-sulfation has long been associated with the modulation of protein-protein interactions in several physiological processes. A number of relevant interactions for hemostasis are largely dictated by this PTM, many of which involving the serine proteinase thrombin (FIIa), a central player in the blood-clotting cascade. Tyrosine sulfation is not limited to endogenous FIIa ligands and has also been found in hirudin, a well-known and potent thrombin inhibitor from the medicinal leech, Hirudo medicinalis. The discovery of hirudin led to successful clinical application of analogs of leech-inspired molecules, but also unveiled several other natural thrombin-directed anticoagulant molecules, many of which undergo tyrosine-O-sulfation. The presence of this PTM has been shown to enhance the anticoagulant properties of these peptides from a range of blood-feeding organisms, including ticks, mosquitos and flies. Interestingly, some of these molecules display mechanisms of action that mimic those of thrombin's bona fide substrates.

A novel metalloproteinase-derived cryptide from Bothrops cotiara venom inhibits angiotensin-converting enzyme activity.

Snake venoms are primarily composed of proteins and peptides, which selectively interact with specific molecular targets, disrupting prey homeostasis. Identifying toxins and the mechanisms involved in envenoming can lead to the discovery of new drugs based on natural peptide scaffolds. In this study, we used mass spectrometry-based peptidomics to sequence 197 peptides in the venom of Bothrops cotiara, including a novel 7-residue peptide derived from a snake venom metalloproteinase. This peptide, named Bc-7a, features a pyroglutamic acid at the N-terminal and a PFR motif at the C-terminal, homologous to bradykinin. Using FRET (fluorescence resonance energy transfer) substrate assays, we demonstrated that Bc-7a strongly inhibits the two domains of angiotensin converting enzyme (Ki < 1 µM). Our findings contribute to the repertoire of biologically active peptides from snake venoms capable of inhibiting angiotensin-converting enzyme (ACE), beyond current known structural motifs and precursors. In summary, we report a novel snake venom peptide with ACE inhibitory activity, suggesting its potential contribution to the hypotensive effect observed in envenomation.

The structural plasticity of polyglutamine repeats.

From yeast to humans, polyglutamine (polyQ) repeat tracts are found frequently in the proteome and are particularly prominent in the activation domains of transcription factors. PolyQ is a polymorphic motif that modulates functional protein-protein interactions and aberrant self-assembly. Expansion of the polyQ repeated sequences beyond critical physiological repeat length thresholds triggers self-assembly and is linked to severe pathological implications. This review provides an overview of the current knowledge on the structures of polyQ tracts in the soluble and aggregated states and discusses the influence of neighboring regions on polyQ secondary structure, aggregation, and fibril morphologies. The influence of the genetic context of the polyQ-encoding trinucleotides is briefly discussed as a challenge for future endeavors in this field.

Crystallization and preliminary crystallographic characterization of three peptidic inhibitors in complex with α-thrombin.

The serine protease thrombin plays a major role in thrombosis and haemostasis. This has driven interest in thrombin inhibitors as potential antithrombotic drugs. Here, the crystallization and preliminary crystallographic analysis of human α-thrombin in complex with three noncovalent peptide inhibitors of the general sequence D-Phe-Pro-D-Arg-P1'-CONH2 are reported. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1) and diffracted to beyond 1.3 Šresolution.

Purification, crystallization and preliminary X-ray diffraction analysis of the seryl-tRNA synthetase from Candida albicans.

The seryl-tRNA synthetase (SerRS) from Candida albicans exists naturally as two isoforms resulting from ambiguity in the natural genetic code. Both enzymes were crystallized by the sitting-drop vapour-diffusion method using 3.2-3.4 M ammonium sulfate as precipitant. The crystals belonged to the hexagonal space group P6(1)22 and contained one monomer per asymmetric unit, despite the synthetase existing as a homodimer (with a molecular weight of ∼116 kDa) in solution. Diffraction data were collected to 2.0 Šresolution at a synchrotron source and the crystal structures of unliganded SerRS and of its complexes with ATP and with a seryl-adenylate analogue were solved by molecular replacement. The structure of C. albicans SerRS represents the first reported structure of a eukaryotic cytoplasmic SerRS.

Human procarboxypeptidase B: three-dimensional structure and implications for thrombin-activatable fibrinolysis inhibitor (TAFI).

Besides their classical role in alimentary protein degradation, zinc-dependant carboxypeptidases also participate in more selective regulatory processes like prohormone and neuropeptide processing or fibrinolysis inhibition in blood plasma. Human pancreatic procarboxypeptidase B (PCPB) is the prototype for those human exopeptidases that cleave off basic C-terminal residues and are secreted as inactive zymogens. One such protein is thrombin-activatable fibrinolysis inhibitor (TAFI), also known as plasma PCPB, which circulates in human plasma as a zymogen bound to plasminogen. The structure of human pancreatic PCPB displays a 95-residue pro-segment consisting of a globular region with an open-sandwich antiparallel-alpha antiparallel-beta topology and a C-terminal alpha-helix, which connects to the enzyme moiety. The latter is a 309-amino acid residue catalytic domain with alpha/beta hydrolase topology and a preformed active site, which is shielded by the globular domain of the pro-segment. The fold of the proenzyme is similar to previously reported procarboxypeptidase structures, also in that the most variable region is the connecting segment that links both globular moieties. However, the empty active site of human procarboxypeptidase B has two alternate conformations in one of the zinc-binding residues, which account for subtle differences in some of the key residues for substrate binding. The reported crystal structure, refined with data to 1.6A resolution, permits in the absence of an experimental structure, accurate homology modelling of TAFI, which may help to explain its properties.

Tick-derived Kunitz-type inhibitors as antihemostatic factors.

Endogenous Kunitz-type inhibitors target a large number of serine proteinases, including coagulation factors VIIa and Xa, but not thrombin. By contrast, several two-domain Kunitz inhibitors of this major procoagulant proteinase have been isolated from both soft ticks (e.g., ornithodorin from Ornithodoros moubata) and hard ticks (e.g., boophilin from Rhipicephalus (Boophilus) microplus). Surprisingly, these anticoagulants do not follow the canonical mechanism of proteinase inhibition. Instead, their N-terminal residues bind across the thrombin active-site cleft, while C-terminal modules interact with the basic exosite I. The reactive-site loop of boophilin remains fully accessible in its complex with thrombin, and might interact with FXa according to the standard mechanism. A conceptually similar inhibition mechanism is employed by a related inhibitor of the TF-FVIIa complex isolated from Ixodes scapularis, ixolaris. Significant variations to the Kunitz fold are encountered in several of these factors, and are particularly evident in the single-domain FXa inhibitor, O. moubata TAP, and in soft tick-derived platelet antiaggregants (e.g., O. moubata disagregin). Altogether, these antihemostatic factors illustrate the divergence between hard and soft ticks. The unsurpassed versatility of tick-derived Kunitz inhibitors establishes them as valuable tools for biochemical investigations, but also as lead compounds for the development of novel antithrombotics.