DP-EP protein crystal structure. a , the protein monomer forms a ␤ sandwich structure and is annotated as per the legend to Fig. 1. Rainbow coloring from red to blue indicates the N- to C-terminal positions of the residues in the model. b , the protein forms dimers in crystals. The monomers are colored separately, as are the C-terminal fragments. 

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Context 1

... simulation model, in which no protein atoms interacted directly with proteins atoms in the periodic images. Electrostatic interactions were computed using a smooth particle mesh Ewald method (21), with a grid size of ϳ 1 Å spacing. Simulations began with a small amount of minimi- zation ( ϳ 1,000 steps) and then utilized typically 100,000 steps of NVT dynamics and 100,000 steps of NPT dynamics run with 1-fs time steps to equilibrate the system. Constant temperature was maintained by a Langevin method (22), and constant pressure conditions were enforced through a modified version of the Langevin piston (23) and Hoover (24, 25) methods. Typical production runs were of 1-ns duration and were conducted using NPT dynamics with 2-fs time steps, recording coordinate information at 1-ps intervals. Simulations were conducted for monomeric, dimeric, and hexameric conformations of the protein, with the majority of simulations utilizing the dimeric form. In these cases, there were ϳ 28,000 atoms in the simulation, with a simulation box size of ϳ 50 ϫ 65 ϫ 80 Å. For the hexameric simulations, the model included ϳ 63,000 atoms in a box of dimensions 95 ϫ 101 ϫ 70 Å. We observed noticeable differences in structure between the monomeric and dimeric forms but no appreciable differences between dimeric and hexameric forms. In the molecular dynamics simulations that began from the native crystal structure, we observed that the initial minimiza- tion steps restored the cleaved peptide bond. Subsequent trajectories were compared with crystal structures of the non- cleaving mutants: the r.m.s. deviation difference between C ␣ atoms of the crystal structure and average simulation structures was generally in the range of 1.5–1.7 Å, with the largest differences arising from mobile loops. The simulations were conducted in a diffuse limit, where protein atoms were not sub- jected to packing forces from other protein atoms, thereby accounting for much of the observed differences in structure. Studies —To study energetics along the proposed reaction pathway, we performed hybrid QM/MM calculations with the program NWChem (26). We utilized a nudged elastic band (NEB) method (27) implemented recently. Initial coordinates for reactant states were taken from snapshots of the dynamics trajectories in which the attacking water molecule was positioned in what we would describe as a near attack conformation (28). The quantum partitions for all simulations included residues 114 –118 and an attacking water (supplemental Fig. S4); hydrogen atoms were used as link atoms. Atoms beyond 15 Å from the target carbonyl carbon atom of Asp-116 were frozen in place, and only atoms within that spherical region were allowed to move. Reactant states were defined by a process in which the original model was optimized using the density func- tional method B3LYP (29) and a 3–21g* basis set to speed con- vergence to a coarse estimate of the state geometry. All subse- quent QM/MM calculations used the 6 –31 ϩϩ g** basis set for all quantum atoms. Product states were constructed by initially constraining the O–C distance between the attacking water and the carbonyl carbon of Asp-116 to 1.5 Å and optimizing the geometry at the B3LYP/6 –31 ϩϩ g** level of theory. One of the water protons was then constrained to be 1 Å from the N nitrogen atom of Pro-117, and, if necessary, the N–C distance between the two leaving groups was constrained to be 3.1 Å. The geometry optimizations were then repeated. All atom constraints were then removed, and the model geometry was reoptimized. The NEB simulations required an initial guess for states along the reaction pathway. These initial guesses were provided by optimizations with constraints applied to key atom distance. In the native protein simulation, for example, the distance between one of the water protons and the O ␦ 2 oxygen atom of Asp-116 was first shortened stepwise in 0.2-Å increments to a final value of 1 Å. The O–C distance between the water oxygen atom and the C carbon atom of Asp-116 was then shortened stepwise in 0.2-Å increments from its initial value to 1.35 Å. The water proton was then moved stepwise to the N nitrogen atom of Pro-117, and finally the C–N distance was pushed out stepwise to 3 Å. This initial guess for the pathway resulted in 29 replicas (beads) of the system. The NEB methodology then was used to define an optimized pathway, one that provided a minimum energy path from reactant to product state. Free energies were obtained by running 20 ps of molecular dynamics to estimate the change in free energy from one bead to the next. The native protein crystallizes readily and rhombohedral crystals diffract to high resolution. The crystal structure was determined by a single-wavelength anomalous diffraction method and refined to 1.57 Å. Crystallographic data are summarized in Table 1. The asymmetric unit of the protein structure contains a single protein monomer, as illustrated in sche- matic form in Fig. 2. The monomer forms a ␤ sandwich consisting of two mixed ␤ sheets. The structure shows distant similarity to convalin (Protein Data Bank entry 1DGR). Unex- pectedly, the polypeptide chain near the C-terminal end of the protein is severed, as can be seen more readily in the electron density depicted in Fig. 3. Two distinct polypeptides are clearly visible at 1.57 Å resolution; the N-terminal fragment includes residues 1–116, and the C-terminal fragment includes residues 117–125. The C-terminal, ␤ 10 strand remains bound to the protein main body in a groove between strands ␤ 3 and ␤ 8 . In addition, the electron density maps clearly revealed that the Lys-98 side chain nitrogen atom is covalently linked to the peptide carbon atom of Asp-116 (Fig. 3). The major multimeric form of the protein in the crystal appears to be a dimer (Fig. 2) formed by two monomers related by 2-fold crystallographic symmetry. The dimerization interface is quite extensive and involves a number of direct and solvent-mediated interactions using main chain and side chain atoms. The total buried sol- vent-accessible area due to the dimer formation is about 1,243 Å (2). In the crystal, three dimers assemble into a hexamer (supplemental Fig. S1). Proteolytic Activity and Oligomerization —We initially hypothesized that the DP-EP protein was an aspartic endopeptidase utilizing the aspartate residue present to cleave the peptide bond. There is a clear pH dependence evident, with an optimal pH between 6 and 6.2. Tests for endopeptidase activity using BSA protein did not show any degradation of BSA. Hence, the SO1698 protein seems to have only autoproteolytic activity. In Fig. 4, we also observe an oligomeric band. The band corresponds to 57 kDa based on used molecular weight standards, which could represent a protein tetramer. However, the protein monomer migrates as a 10.5-kDa protein in the same gel instead of a 13.7 kDa band, and the cleaved monomer migrates as a 9-kDa protein instead of a 12.7 kDa band. This anomaly could be explained by a low protein pI value equal to 4.23. It has been observed that acidic proteins migrate on SDS-PAGE anomalously. Thus, the observed cross-linked band represents protein hexamer based on SDS-polyacrylamide gels (expected molecular mass based on migration of cleaved monomer, is 54 kDa). The oligomerization state of SO1698 in solution was further investigated by size exclusion chromatography. Under native conditions, only one major chromatography fraction is observed. This faction corresponds to a 108-kDa homo-oli- gomer (supplemental Fig. S2). The SO1698 D37A mutant migrates with similar apparent molecular mass (104 kDa). In solution, the apparent molecular mass of SO1698 is larger than hexamers (82.2 kDa) observed in crystals. However, because the SO1698 is donut-shaped with dimensions of 80 ϫ 45 Å, it may appear larger than globular proteins. Therefore, we conclude that SO1698 is a hexamer both in the crystal and in solution. Formation of the oligomer is also pH-dependent, forming only at acidic pH. The unusual stability of oligomers under denaturing conditions of SDS-PAGE could be explained by covalent bonding of hexamers; however, we did not observe any such connections in our structure. The two N-terminal residues are not visible in the crystal structure, so we tested truncation mutants of 4, 7, and 10 N-terminal residues to exclude the possibility that these residues participate in the covalent linkage. Each of these mutants exhibited biochemical proper- ties and crystallized in the same fashion as wild-type protein (data not shown). Additionally, we tested multiple crystals of DP-EP protein crystallizing in different morphological forms and having different space groups. All displayed the same dimeric and hexameric assemblies. Active Site Mutagenesis —To analyze the autoproteolytic activity of the protein, we constructed eight catalytic site mutants: Y26F, D37A, K98A, S114A, P115A, D116A, P117A, and Q118A. All mutants were crystallized at various pH conditions, and their structures were determined at high resolution (range 1.25–2.45 Å). A summary of the crystallographic data is provided in supplemental Table S1. The crystal structures were analyzed for presence/absence of a bond between residues 116 and 117. A summary of the mutagenesis analysis is provided in Table 2. Two mutations (D116A and P117A) resulted in com- plete loss of autoproteolytic function, and one mutation (D37A) resulted in major loss of function. In addition, the S114A mutation displayed substantial reduction of the activity. The remaining mutations had no visible impact on self-cleavage. These results suggest that the two aspartic acid residues (Asp-37 and Asp-116) may be involved in the self-cleaving reaction, as found in peptidases, where two acidic residues are utilized in the catalytic process (30 –32). What we observe in the crystal structures contradicts this assumption. We suppose that the D116A structure ...

Context 2

... dimeric, and hexameric conformations of the protein, with the majority of simulations utilizing the dimeric form. In these cases, there were ϳ 28,000 atoms in the simulation, with a simulation box size of ϳ 50 ϫ 65 ϫ 80 Å. For the hexameric simulations, the model included ϳ 63,000 atoms in a box of dimensions 95 ϫ 101 ϫ 70 Å. We observed noticeable differences in structure between the monomeric and dimeric forms but no appreciable differences between dimeric and hexameric forms. In the molecular dynamics simulations that began from the native crystal structure, we observed that the initial minimiza- tion steps restored the cleaved peptide bond. Subsequent trajectories were compared with crystal structures of the non- cleaving mutants: the r.m.s. deviation difference between C ␣ atoms of the crystal structure and average simulation structures was generally in the range of 1.5–1.7 Å, with the largest differences arising from mobile loops. The simulations were conducted in a diffuse limit, where protein atoms were not sub- jected to packing forces from other protein atoms, thereby accounting for much of the observed differences in structure. Studies —To study energetics along the proposed reaction pathway, we performed hybrid QM/MM calculations with the program NWChem (26). We utilized a nudged elastic band (NEB) method (27) implemented recently. Initial coordinates for reactant states were taken from snapshots of the dynamics trajectories in which the attacking water molecule was positioned in what we would describe as a near attack conformation (28). The quantum partitions for all simulations included residues 114 –118 and an attacking water (supplemental Fig. S4); hydrogen atoms were used as link atoms. Atoms beyond 15 Å from the target carbonyl carbon atom of Asp-116 were frozen in place, and only atoms within that spherical region were allowed to move. Reactant states were defined by a process in which the original model was optimized using the density func- tional method B3LYP (29) and a 3–21g* basis set to speed con- vergence to a coarse estimate of the state geometry. All subse- quent QM/MM calculations used the 6 –31 ϩϩ g** basis set for all quantum atoms. Product states were constructed by initially constraining the O–C distance between the attacking water and the carbonyl carbon of Asp-116 to 1.5 Å and optimizing the geometry at the B3LYP/6 –31 ϩϩ g** level of theory. One of the water protons was then constrained to be 1 Å from the N nitrogen atom of Pro-117, and, if necessary, the N–C distance between the two leaving groups was constrained to be 3.1 Å. The geometry optimizations were then repeated. All atom constraints were then removed, and the model geometry was reoptimized. The NEB simulations required an initial guess for states along the reaction pathway. These initial guesses were provided by optimizations with constraints applied to key atom distance. In the native protein simulation, for example, the distance between one of the water protons and the O ␦ 2 oxygen atom of Asp-116 was first shortened stepwise in 0.2-Å increments to a final value of 1 Å. The O–C distance between the water oxygen atom and the C carbon atom of Asp-116 was then shortened stepwise in 0.2-Å increments from its initial value to 1.35 Å. The water proton was then moved stepwise to the N nitrogen atom of Pro-117, and finally the C–N distance was pushed out stepwise to 3 Å. This initial guess for the pathway resulted in 29 replicas (beads) of the system. The NEB methodology then was used to define an optimized pathway, one that provided a minimum energy path from reactant to product state. Free energies were obtained by running 20 ps of molecular dynamics to estimate the change in free energy from one bead to the next. The native protein crystallizes readily and rhombohedral crystals diffract to high resolution. The crystal structure was determined by a single-wavelength anomalous diffraction method and refined to 1.57 Å. Crystallographic data are summarized in Table 1. The asymmetric unit of the protein structure contains a single protein monomer, as illustrated in sche- matic form in Fig. 2. The monomer forms a ␤ sandwich consisting of two mixed ␤ sheets. The structure shows distant similarity to convalin (Protein Data Bank entry 1DGR). Unex- pectedly, the polypeptide chain near the C-terminal end of the protein is severed, as can be seen more readily in the electron density depicted in Fig. 3. Two distinct polypeptides are clearly visible at 1.57 Å resolution; the N-terminal fragment includes residues 1–116, and the C-terminal fragment includes residues 117–125. The C-terminal, ␤ 10 strand remains bound to the protein main body in a groove between strands ␤ 3 and ␤ 8 . In addition, the electron density maps clearly revealed that the Lys-98 side chain nitrogen atom is covalently linked to the peptide carbon atom of Asp-116 (Fig. 3). The major multimeric form of the protein in the crystal appears to be a dimer (Fig. 2) formed by two monomers related by 2-fold crystallographic symmetry. The dimerization interface is quite extensive and involves a number of direct and solvent-mediated interactions using main chain and side chain atoms. The total buried sol- vent-accessible area due to the dimer formation is about 1,243 Å (2). In the crystal, three dimers assemble into a hexamer (supplemental Fig. S1). Proteolytic Activity and Oligomerization —We initially hypothesized that the DP-EP protein was an aspartic endopeptidase utilizing the aspartate residue present to cleave the peptide bond. There is a clear pH dependence evident, with an optimal pH between 6 and 6.2. Tests for endopeptidase activity using BSA protein did not show any degradation of BSA. Hence, the SO1698 protein seems to have only autoproteolytic activity. In Fig. 4, we also observe an oligomeric band. The band corresponds to 57 kDa based on used molecular weight standards, which could represent a protein tetramer. However, the protein monomer migrates as a 10.5-kDa protein in the same gel instead of a 13.7 kDa band, and the cleaved monomer migrates as a 9-kDa protein instead of a 12.7 kDa band. This anomaly could be explained by a low protein pI value equal to 4.23. It has been observed that acidic proteins migrate on SDS-PAGE anomalously. Thus, the observed cross-linked band represents protein hexamer based on SDS-polyacrylamide gels (expected molecular mass based on migration of cleaved monomer, is 54 kDa). The oligomerization state of SO1698 in solution was further investigated by size exclusion chromatography. Under native conditions, only one major chromatography fraction is observed. This faction corresponds to a 108-kDa homo-oli- gomer (supplemental Fig. S2). The SO1698 D37A mutant migrates with similar apparent molecular mass (104 kDa). In solution, the apparent molecular mass of SO1698 is larger than hexamers (82.2 kDa) observed in crystals. However, because the SO1698 is donut-shaped with dimensions of 80 ϫ 45 Å, it may appear larger than globular proteins. Therefore, we conclude that SO1698 is a hexamer both in the crystal and in solution. Formation of the oligomer is also pH-dependent, forming only at acidic pH. The unusual stability of oligomers under denaturing conditions of SDS-PAGE could be explained by covalent bonding of hexamers; however, we did not observe any such connections in our structure. The two N-terminal residues are not visible in the crystal structure, so we tested truncation mutants of 4, 7, and 10 N-terminal residues to exclude the possibility that these residues participate in the covalent linkage. Each of these mutants exhibited biochemical proper- ties and crystallized in the same fashion as wild-type protein (data not shown). Additionally, we tested multiple crystals of DP-EP protein crystallizing in different morphological forms and having different space groups. All displayed the same dimeric and hexameric assemblies. Active Site Mutagenesis —To analyze the autoproteolytic activity of the protein, we constructed eight catalytic site mutants: Y26F, D37A, K98A, S114A, P115A, D116A, P117A, and Q118A. All mutants were crystallized at various pH conditions, and their structures were determined at high resolution (range 1.25–2.45 Å). A summary of the crystallographic data is provided in supplemental Table S1. The crystal structures were analyzed for presence/absence of a bond between residues 116 and 117. A summary of the mutagenesis analysis is provided in Table 2. Two mutations (D116A and P117A) resulted in com- plete loss of autoproteolytic function, and one mutation (D37A) resulted in major loss of function. In addition, the S114A mutation displayed substantial reduction of the activity. The remaining mutations had no visible impact on self-cleavage. These results suggest that the two aspartic acid residues (Asp-37 and Asp-116) may be involved in the self-cleaving reaction, as found in peptidases, where two acidic residues are utilized in the catalytic process (30 –32). What we observe in the crystal structures contradicts this assumption. We suppose that the D116A structure represents the conformational state of the active site of the native protein less the catalytic carboxyl moiety. In the D116A structure, the carboxyl group of Asp-37 is ϳ 6 Å from the amide carbon atom of Ala-116; this distance is seemingly too large for the carboxyl group of Asp-37 to play a direct role in the self-cleaving function of the protein. To understand how loss of the carboxyl group from Asp-37 leads to loss of self-cleaving function in the D37A mutant, we compare the D116A and D37A structures in Fig. 5. We observe that the mutation causes a significant reorganization of the active site. The protein backbones were aligned on the C atoms from residue 60 to 100 (r.m.s. deviation ϭ 1.16 Å). There is a register shift in the ␤ strands in the D37A structure. In the alignment depicted in Fig. 5, the Gln-118 side chain has rotated from ...