Structural and Functional Basis of Plasminogen Activation by Staphylokinase

Abstract

SummaryStaphylokinase (Sak), a 15.5-kDa bacterial protein, forms a complex with human plasmin, which in turn activates other plasminogen molecules to plasmin. Three recombinant DNA-based approaches, (i) site directed substitution with alanine, (ii) search for proximity relationships at the complex interface, and (iii) active-site accessibility to protease inhibitors have been used to deduce a coherent docking model of the crystal structure of Sak on the homology-based model of micro-plasmin (μPli), the serine protease domain of plasmin. Sak binding on μPli is primarily mediated by two surface-exposed loops, loops 174 and 215, at the rim of the active-site cleft, while the binding epitope of Sak on μPli involves several residues located in the flexible NH2-terminal arm and in the five-stranded mixed β-sheet. Several Sak residues located within the unique μ-helix and the β2 strand do not contribute to the binding epitope but are essential to induce plasminogen activating potential in the Sak:μPli complex. These residues form a topologically distinct activation epitope, which, upon binding of Sak to the catalytic domain of μPli, protrudes into a broad groove near the catalytic triad of μPli, thereby generating a competent binding pocket for micro-plasminogen (μPlg), which buries approximately 2500 Å of the Sak:μPli complex upon binding. This structural and functional model may serve as a template for the design of improved Sak-derived thrombolytic agents. Following the completion and presentation of the present study, the deduced Sak:μPli:μPlg complex was fully confirmed by X-ray crystallography, which further illustrates the power and potential of the present approach.

Structural Consideration of the Formation of the Activation Complex between the Staphylokinase-Like Streptococcal Plasminogen Activator PadA and Bovine Plasminogen

“…Both were placed in close proximity to the Plm active centre residues in positions consistent with their ability to inhibit interactions with PadA, leading to complex formation. Residues identified as performing critical roles in the interaction of human microplasminogen (hμPlg) with SAK (Arg175 and Arg94) 16,59,60 are located in two surfaceexposed loops (shunt 94 and loop 174) that border the active site region. The P32 peptide sequence from bμPlg maps to a position midway between the structural equivalent of these loops (Fig.…”

Rat cerebral ischemia induced with photochemical occlusion of proximal middle cerebral artery: a stroke model for MR imaging research

“…rCBV, rCBF) changes, (2) to predict final infarct size with PWI at an early time point after stroke onset, (3) to filter an appropriate individual PIT model with penumbra and to evaluate the therapeutic effects of new thrombolytic agents, such as Microplasmin [21]. From the outcomes of the present experiment with a clinical magnet, a positive perspective would be that the introduced proximal PIT stroke model can be equally feasible, if not more, for extensive stroke studies using dedicated high-field-strength magnets in small animals.…”

Visualization of Stroke with Clinical MR Imagers in Rats: A Feasibility Study

“…The photochemically induced thrombosis model of proximal MCA occlusion is believed to be advantageous over other models in terms of its possibilities in both permanent ischemia and postischemic reperfusion and is therefore ideal for studies of new antistroke medications (16,20,21), such as thrombolytic microplasmin (22). With conventional invasive autopsy techniques, however, a large number of animals is required, since only limited information can be obtained from one sacrificed animal at a single time point.…”