Table 3. Interactions of the VEEV nsP2pro active site residues with a peptide substrate and the N terminus of the protease domain.

The interactions at the active site are shown for the binding of the residues of an oligopeptide substrate or the N terminus (“dyn-inact” structures). The hydrophobic contacts and the H-bonds are also included in the table. The residues of the binding sites forming H-bonds with those of the substrate are underlined. The values in parentheses indicate the number of observed H-bonds. The hydrophobic interactions were mapped based on a modeled enzyme-substrate complex and on the cluster representative structure for the self-inactivated conformations extracted from the simulations. The LigPlot+²⁴ software was used to map the hydrophobic interactions.

Binding site residues *	Substrate residue	Enzyme-substrate complex **	N-terminal residue (8DUF / 6BCM)	8DUFdyn-inact	6BCMdyn-inact
-	P6-Leu1	S511, E513, I542	- / Q471	-	-
-	P5-Gln2	I514, I698, S701, A728, I732	A472 / N472	M702	I514, I698, S701, A728, S731, I732
S511, E513, W547, M702, K706*	P4-Glu3	H510, S511, W547, I698, S701, M702, K706	K473 / K473	I542, W547	S511, I542, W547
H510, I542, W547, I698, M702	P3-Ala4	A509, H510, I542, D664, I698, M702	A474 / A474	A509, H510, D664, I698, M702	A509, H510, D664, M701
W478, C477, A509, H510, W547	P2-Gly5	W478, A509, H510, N545, W547	N475 / A475	A472, K473, A509,	A509, H510 (2), I542, N545, H546, W547
				H510 (2), I542, N545, W547
N475, C477, W478, A509, N545, H546, L665	P1-Ala6	C477, W478, N545, H546, W547	V476 / V476	N545, H546, W547	N545, H546, W547
A474, N475, C477, K480, H546	P1'-Gly7	N475, C477,	C477 / C477	A479, K480, A481, H546	A479, K480, A481, H546, W547
		R662 (2)	W478 / W478	K480, L482, F504, K508, H510, W547, V615	A479, K480, A481, L482, V501, D502, Y503, F504, K508

^*The substrate binding site-forming residues have been determined previously based on the complex of VEEV nsP2pro and a substrate representing P4-P1' residues of nsP12 cleavage site (EAGAG).²³

^**The enzyme-substrate interactions were mapped based on a modeled complex of VEEV nsP2 protease and LQEAGAjGSVETP substrate.¹⁴ The coordinate file was kindly provided previously¹⁵ by Patricia M. Legler (Center for Bio/molecular Science and Engineering, U.S. Naval Research Laboratory).