First crystal structure of double knotted protein TrmD-Tm1570 – inside from degradation perspective

Herein, we present the first crystal structure of a double knotted protein TrmD-Tm1570 from Calditerrivibrio nitroreducens , as well the X-ray structure of each sub-domain. The protein consists of two domains TrmD and Tm1570, each embedding a single trefoil knot, which can function on their own. TrmD-Tm1570 forms a compact homodimeric complex. This protein represents one of 296 possible doubly knotted proteins from SPOUT family. Based on TrmD-Tm1570 from Calditerrivibrio nitroreducens we show that a double knotted protein can be fully degraded by the ClpXP degradation system, as well as its individual domains. We used numerical simulations to explain the difference in the speed of degradation. The derived kinetic parameters for the degradation process are comparable to the experimental data found for unknotted polypeptide chains.