calculation report
FMO calculations for zinc metalloprotease:Fragmentation of amino-acid residues coordinated to zinc ion
https://ror.org/023rffy11 
2022 年 22 巻 p. 21-25
詳細
2022 年 22 巻 p. 21-25
We investigated electronic states of a complex of zinc metalloprotease ubiquitin ligase 2(UBR2) with its peptide ligand using ab initio fragment molecular orbital (FMO) calculations. UBR2 possesses three Zn ions and several residues of UBR2 are coordinated to each Zn ion to form an active site of UBR2. To provide a precise description of these coordination bonds, we included these residues in the same fragment as Zn ion in FMO calculations. The results revealed that all coordinated residues should be included in the same fragment as Zn ion for obtaining the converged results. This fact can be applicable equally to metalloproteases including other metal ions.
We here employed the high-resolution X-ray crystal structure (PDB ID: 5TDA) [1] for a complex of ubiquitin ligase 2 (UBR2) with its peptide ligand composed of Arg-Leu-Trp-Ser residues (RLWS-N-degron) as shown in Figure 1a. The peptide possesses NH3+ and COO− groups at the N- and C-terminals, respectively, and a specific crystal water molecule bridges between the peptide and UBR2. Since the positions of hydrogen atoms of UBR2 and peptide were determined through the experiment [1], we added hydrogen atoms to crystal water molecules and optimized their positions in solvating water molecules using classical molecular mechanics (MM) method of AMBER18 [2]. The charge parameters of the peptide were determined by the restrained electrostatic potential (RESP) analysis of Gaussian, while Zn ions were considered to have +2 charge. In the MM optimizations, the FF14SB and general AMBER force fields, and TIP3P model were assigned to UBR2, peptide, and water molecules, respectively. We added the solvating water molecules with a layer of 8 Å around the complex. To neutralize the charge of the N- and C-terminals of UBR2, they were capped by ACE and NME, respectively, and the structures of the capped groups were optimized by MM method.
FMO calculations were conducted using MP2/6-31G or MP2/6-31G* method [3] of the FMO calculation program ABINIT-MP rev.22 to accurately investigate the π–π stacking, NH–π and CH–π interactions as well as the hydrogen bonding and electrostatic interactions between UBR2 and the peptide ligand. Zn ions were considered to have +2 charge and a singlet spin state. As shown in Figures 1b and 1c, since UBR2 possesses three Zn ions and several residues of UBR2 coordinate strongly to the Zn ions to form an active site of UBR2, these coordination bonds should be precisely described in the FMO calculations. Accordingly, we assigned these residues as the same fragment as the Zn ion. Notably, as shown in Figure 1b, the S atom of Cys127 bridges two Zn ions as Zn1 and Zn2, so that they can exist close to each other. These specific interactions around the two Zn ions seem to be essential for forming the active site of UBR2. We thus considered two patterns of fragmentations and conducted the FMO calculations. In contrast, since the other Zn ion as Zn3 is coordinated to four residues as shown in Figure 1c, these residues were included in the same fragment as the Zn3 ion. The details of the fragmentations are listed in Table 1. The four residues-peptide was treated as a single fragment.
Additionally, to shorten the computational time of the fragment dimer calculations in FMO, we changed the Ldimer parameter (from 2.0 to 1.5) for the ES approximation in FMO and investigated the effect on computational time and inter fragment interaction energies (IFIEs) between the peptide and UBR2 residues. The results were obtained by use of the supercomputer Fugaku of RIKEN.
Figure 1. Structure of complex between UBR2 and RLWS N-degron
Entire structure of the complex (a). Coordinated structure between Zn ions and UBR2 residues; (b) Zn1, Zn2 and seven residues, (c) Zn3 and four residues.
Table 1. Fragmentations of Zn groups for UBR2 protein
In the combined fragmentation, Zn1* and Zn2* are considered as the same fragment.
| Fragment | UBR2 residues |
|---|---|
| Zn1* | Zn1, Cys99, Cys124, Cys127, Cys149 |
| Zn2* | Zn2, Cys151, Cys163, Hie166 |
| Zn3* | Zn3, Cys112, Cys115, Hie133, Hie136 |
| Combined | Zn1, Zn2, Cys99, Cys124, Cys127, Cys149, Cys151, Cys163, Hie166 |
Table 2 summarizes the results on convergence of the present FMO calculations. Only in the case of using the 6-31G basis set and the combined fragmentation, the converged result was obtained. When the 6-31G* basis set was used, it was not possible to obtain the result within 72 hours using 64 nodes (3072 cores) of Fugaku. In contrast, when the fragments Zn1* and Zn2* were considered separately, the SCC or SCF calculation for fragment monomer was not converged. This result might be related to the separation of the coordination bond between the S atom of Cys127 and Zn2 ion shown in Figure 1b. This S atom plays an essential role of bridging the two Zn ions by coordination bonds. Consequently, the present FMO calculations elucidate that the UBR2 residues coordinated to Zn ion should be included in the same fragment as the Zn ion. For such a large fragment containing two Zn ions and seven residues shown in Figure 1b, 6-31G* basis set is too large to obtain a converged result within a normal CPU time and memory. It is therefore concluded that the 6-31G basis set is suitable for obtaining the converged results for the present complex within a realistic time frame.
Table 2. Convergence of FMO calculations for UBR2 + peptide complex
| Basis set | 6-31G | 6-31G | 6-31G* | 6-31G* |
|---|---|---|---|---|
| Fragmentation of Zn1* and Zn2* groups | Combined | Separate | Combined | Separate |
| Number of basis functions of the largest fragment containing Zn | 621 | 405 | 959 | 631 |
| Result | Converged | Not converged*1 | Not finished*2 | Not converged*3 |
*1: Not converged at Monomer SCC *2: Not finished within 72 hours, when proc=64, node=64, NUM_THREAD=2 is used. *3: Not converged at Monomer SCF
To shorten the computational time of FMO calculations for fragment dimers, we changed the Ldimer parameter from 2.0 to 1.5 and investigated the effect on computational time and IFIEs between the peptide and UBR2 residues. The FMO computational time was 5.1 (Ldimer = 2.0) and 5.3 h (Ldimer = 1.5), respectively, indicating that the computational time is not shortened significantly by changing the Ldimer parameter. The evaluated IFIEs between the peptide and each UBR2 residue are listed in Table 3, in which only the residues with more than 0.01 kcal/mol difference in IFIEs are listed. Although total IFIE is 0.7 kcal/mol changed, each IFIE changes at most 0.3 kcal/mol. Therefore, for the UBR2 + peptide complex, the effect of changing the Ldimer parameter is not so significant. Here, the converged FMO calculation data with Ldimer values as 2.0 and 1.5 are registered in the FMO database [4, 5] with its codes (FMODB IDs) as 7G2JK and M3VMZ, respectively.
Figure 2 shows the IFIEs between the peptide and each UBR2 residue evaluated by the MP2/6-31G method with the combined fragmentation and Ldimer of 2.0. Asp150, Asp153, Asp118, and Zn1*+Zn2* group have strong attractive interactions with the peptide. In particular, Asp150 interacts strongly with the peptide. These strong interactions come mainly from the electrostatic interactions between the negatively charged Asp residue and the positively charged Arg residue of the peptide. It was also found that the specific water molecule interacts strongly (−25 kcal/mol) with both the Arg and Asp118 of UBR2 to act effectively as a molecule bridging between the peptide and UBR2.
Table 3. Total IFIE (kcal/mol) and IFIE between UBR2 residue and peptide evaluated by MP2/6-31G (Ldimer is 2.0 or 1.5)
Residues or water with IFIE difference larger than 0.01 kcal/mol are listed.
| Ldimer | 2.0 | 1.5 | Difference |
|---|---|---|---|
| Total IFIE | −627.20 | −626.50 | −0.70 |
| Ser112 | −4.93 | −4.92 | −0.01 |
| Asp118 | −68.18 | −67.87 | −0.31 |
| Leu125 | −8.84 | −8.73 | −0.10 |
| Thr137 | 2.05 | 2.27 | −0.22 |
| Thr148 | −0.77 | −0.73 | −0.04 |
| Glu149 | −36.29 | −36.17 | −0.12 |
| Wat327 | 4.26 | 4.31 | −0.05 |
Figure 2. IFIEs between UBR2 residue and peptide evaluated by FMO (MP2/6-31G, Ldimer is 2.0)
Red bars indicate residues with IFIE larger than −50 kcal/mol.
This research was performed in the activities of the FMO drug design consortium. The authors thank Prof. Yuji Mochizuki of Rikkyo University, and Dr. Tatsuya Nakano and Dr. Yoshio Okiyama of the National Institute of Health Sciences for providing the Fugaku version of ABINIT-MP and general discussions related to FMO calculations. We also acknowledge Dr. Teruki Honma, Dr. Daisuke Takaya and Dr. Kikuko Kamisaka of RIKEN for useful comments and technical support of FMODB registration. The FMO calculations were performed using Fugaku of RIKEN (project ID: hp210130). This study was partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS) from the Japan Agency for Medical Research and Development) (grant number JP21am0101113). CW acknowledges JST PRESTO grant (JPMJPR18GD).
