Analysis of the interactions observed in four domains of the wt NP and R361A mutant.

Loop 1 (residues 73–90), loop2 (residues 200–211), β-sheet 1 (residues 91–112), and the linker (residues 360–373).These interactions define a path between loop 1 and the residue R361 of the linker, located in the RNA binding groove (see Figures 2 and 4).1HB stands for hydrogen bond.2HPh stands for hydrophobic interaction.See the Experimental section for definitions of the contact domains and the two contact types.a)Loop 1-loop 2 contact: In R361A, hydrophobic interactions between L79 and loop 2 drove loop 1 to contact loop2; transient salt-bridges between R204 or R208, on the one hand, and E80 or E81, on the other hand, stabilized the interaction between loop 1 and loop 2 at short distances. Such loop-loop interactions were not found in wt NP.b)Loop 1 stability (base): The side-chain of K113 was engaged in a strong hydrogen bond with the C-terminus of the loop 1; the guanidinium moiety of K113 formed a salt bridge with E73 at the N-terminus of loop 1, contributing to the stability of loop 1 in wt NP. The R361A mutation drastically reduced the interactions of the K113 with the loop 1, increasing loop 1 flexibility.c)Linker-β sheet 1 contacts: The linker was connected to the β sheet 1 through conserved hydrophobic interaction between M371 and the ring of Y97, stable hydrogen bonds between R106 and the linker backbone oxygen atoms. The R361A mutation decreased the population of the solvated K103-E372 salt-bridge.d)Inter-linker contacts: In NP, E369 interacted with both R361 and R317. In R361A, the R317-E369 contact population increased to 93% as compared to 73% in wt NP and the R361-E369 interaction was canceled by the mutation.环1（残基73–90）、环2（残基200–211）、β折叠1（β-sheet 1，残基91–112）以及连接子（残基360–373）。这些相互作用定义了环1与连接子的R361残基之间的通路，该通路位于RNA结合槽（RNA binding groove）内（见图2和图4）。1HB代表氢键（hydrogen bond），2HPh代表疏水相互作用（hydrophobic interaction）。有关接触结构域与两种接触类型的定义，请参见实验部分。 a) 环1-环2接触：在R361A突变体中，L79与环2之间的疏水相互作用促使环1与环2发生接触；一方面，R204或R208，另一方面，E80或E81之间形成的瞬时盐桥，可在短距离内稳定环1与环2之间的相互作用。此类环-环相互作用在野生型NP中未被观察到。 b) 环1稳定性（基础）：K113的侧链与环1的C端形成强氢键；K113的胍基与环1N端的E73形成盐桥，有助于维持野生型NP中环1的稳定性。R361A突变大幅削弱了K113与环1之间的相互作用，提升了环1的柔性。 c) 连接子-β折叠1接触：连接子通过M371与Y97芳环之间的保守疏水相互作用、R106与连接子主链氧原子之间的稳定氢键，与β折叠1相连。R361A突变降低了溶剂化的K103-E372盐桥的占比。 d) 连接子间接触：在NP中，E369可与R361和R317同时发生相互作用。在R361A突变体中，R317-E369的接触占比从野生型NP的73%提升至93%，而R361-E369的相互作用则因该突变被消除。