Dynamically-Driven Inactivation of the Catalytic Machinery of the SARS 3C-Like Protease by the N214A Mutation on the Extra Domain

Despite utilizing the same chymotrypsin fold to host the catalytic machinery, coronavirus 3C-like proteases (3CLpro) noticeably differ from picornavirus 3C proteases in acquiring an extra helical domain in evolution. Previously, the extra domain was demonstrated to regulate the catalysis of the SARS-CoV 3CLpro by controlling its dimerization. Here, we studied N214A, another mutant with only a doubled dissociation constant but significantly abolished activity. Unexpectedly, N214A still adopts the dimeric structure almost identical to that of the wild-type (WT) enzyme. Thus, we conducted 30-ns molecular dynamics (MD) simulations for N214A, WT, and R298A which we previously characterized to be a monomer with the collapsed catalytic machinery. Remarkably, three proteases display distinctive dynamical behaviors. While in WT, the catalytic machinery stably retains in the activated state; in R298A it remains largely collapsed in the inactivated state, thus implying that two states are not only structurally very distinguishable but also dynamically well separated. Surprisingly, in N214A the catalytic dyad becomes dynamically unstable and many residues constituting the catalytic machinery jump to sample the conformations highly resembling those of R298A. Therefore, the N214A mutation appears to trigger the dramatic change of the enzyme dynamics in the context of the dimeric form which ultimately inactivates the catalytic machinery. The present MD simulations represent the longest reported so far for the SARS-CoV 3CLpro, unveiling that its catalysis is critically dependent on the dynamics, which can be amazingly modulated by the extra domain. Consequently, mediating the dynamics may offer a potential avenue to inhibit the SARS-CoV 3CLpro

Full-endoscopic technique for anterior cervical discectomy and interbody fusion: 5-year follow-up results of 67 cases

With minimally invasive technique becoming more popular, endoscopic operations such as arthroscopy or laparoscopy have become the standard of care in several other areas. In this study, we evaluated the 5-year follow-up outcomes of anterior cervical (Ahn et al. in Photomed Laser Surg 23:362–368, 2005) discectomy and interbody fusion (ACDF) performed via endoscopic approach. Sixty-seven patients who underwent anterior cervical discectomy and cage fusion performed using endoscopic technique were followed for at least 5 years. We reviewed the clinical and radiographic records of these patients. The postoperative radiographic measures accessed were the anterior intervertebral height (AIH) and the lordosis angle (LDA). Clinical outcomes were determined using the previously validated Japanese Orthopaedic Association (JOA) and the pain visual analog scale (VAS). Patients included had a minimal follow-up period of 5 years and based on the outcomes criteria (JOA, VAS), 86.6% of patients reported excellent or good results. The AIH increased on average 18.7% of the original height (p < 0.01), and the LDA were more physiologic at final follow-up. Of the 67 cases, there was no segmental instability, and the bone fusion rate was 100%. One patient required revision open ACDF due to adjacent segment disc herniation 6 years postoperatively. There were no intraoperative complications, dysphasia or esophageal injury in this study group. It indicated endoscopic technique for ACDF can obtain satisfactory results in patients with cervical disc herniation, cervical myelopathy, or radiculopathy. Compared with a traditional approach, this technique may be associated with less morbidity while improving cosmesis and postoperative recovery. Prospective randomized control trials are needed to directly compare these two procedures