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Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop).

Engineered point mutations in loop 1 (E409K and G410K) are shown to destabilise the dimer interface, lead to a higher proportion of the bound monomer and, in NF-κB luciferase reporter assays, are associated with reduced NF-κB activity compared with wt-p62.

The Vλ VI proteins accumulate non-conservative mutations in loop C–C' (part of CDR2) with 83% of patients with mutations in this region and presenting an absence of non-conservative mutations in loop A B and loop D E (structural representation of differences in Vλ is shown in Figure 4).

Mutations in loop 3 severely compromise the GlpG activity, suggesting the interaction between the substrate and loop 3 is functionally important (Baker and Urban, 2012).

(D ) Different combinations of remote mutations in loop 3 resulted in identical positioning of Leu 106, Trp 131, and Leu 132 in wt PTE and neo PTE (see also Figure 2 figure supplement 1C ).

In the present study, the presence of TP53 mutations in loop 2 and loop 3 did not seem to influence the disease-free survival on early-stage disease, although a 17% difference in the 3-year survival rate was seen.

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(E ) Rewiring the interaction network in neo PTE by remote mutations in loops 4 and 5 led to β-metal displacement (see also Figure 2 figure supplement 1D ).

Fourth, the distance between the two active site zinc ions decreased from 3.8 to 3.3 Å in the forward evolution through displacement of the metal-chelating His201 and the β-metal, which was likely triggered by the combined action of several remote mutations in loops 4 and 5 (t172I, q180H, t199I, and a204G, Figure 2).

POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C).

The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating.

Most mutations occurred in loop regions.

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