Revealing the Origin of the Efficiency of the De Novo Designed Kemp Eliminase HG-3.17 by Comparison with the Former Developed HG-3

The design of new biocatalysts is a goal in biotechnology to improve the rate, selectivity and environmental impact of industrial chemical processes. In this regard, the use of computational techniques has provided valuable assistance in the design of new enzymes with remarkable catalytic activity. In this paper, hybrid QM/MM molecular dynamics simulations have allowed insights to be gained on the origin of the limited efficiency of a computationally designed enzyme for the Kemp elimination; the HG-3. Comparison of results derived from this enzyme with those of a more evolved protein containing additional point mutations, HG-3.17, rendered important information that should be taken into account in the design of new enzymes. For this Kemp eliminase reaction, higher reactivity has been demonstrated to be related to a better electrostatic preorganisation of an environment that creates a more favourable electrostatic potential for the reaction to proceed. The limitations of HG-3 can be related to a lack of flexibility, a not well-fitted active site, and a lack of protein electrostatic preorganisation, which decrease the reorganisation around the oxyanion hole

The evolution of multiple active site configurations in a designed enzyme

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis

Revealing the Origin of the Efficiency of the De Novo Designed Kemp Eliminase HG-3.17 by Comparison with the Former Developed HG-3

The design of new biocatalysts is a goal in biotechnology to improve the rate, selectivity and environmental impact of industrial chemical processes. In this regard, the use of computational techniques has provided valuable assistance in the design of new enzymes with remarkable catalytic activity. In this paper, hybrid QM/MM molecular dynamics simulations have allowed insights to be gained on the origin of the limited efficiency of a computationally designed enzyme for the Kemp elimination; the HG-3. Comparison of results derived from this enzyme with those of a more evolved protein containing additional point mutations, HG-3.17, rendered important information that should be taken into account in the design of new enzymes. For this Kemp eliminase reaction, higher reactivity has been demonstrated to be related to a better electrostatic preorganisation of an environment that creates a more favourable electrostatic potential for the reaction to proceed. The limitations of HG-3 can be related to a lack of flexibility, a not well-fitted active site, and a lack of protein electrostatic preorganisation, which decrease the reorganisation around the oxyanion hole

Monovalent engagement of the BCR activates ovalbumin-specific transnuclear B cells

Valency requirements for B cell activation upon antigen encounter are poorly understood. OB1 transnuclear B cells express an IgG1 B cell receptor (BCR) specific for ovalbumin (OVA), the epitope of which can be mimicked using short synthetic peptides to allow antigen-specific engagement of the BCR. By altering length and valency of epitope-bearing synthetic peptides, we examined the properties of ligands required for optimal OB1 B cell activation. Monovalent engagement of the BCR with an epitope-bearing 17-mer synthetic peptide readily activated OB1 B cells. Dimers of the minimal peptide epitope oriented in an N to N configuration were more stimulatory than their C to C counterparts. Although shorter length correlated with less activation, a monomeric 8-mer peptide epitope behaved as a weak agonist that blocked responses to cell-bound peptide antigen, a blockade which could not be reversed by CD40 ligation. The 8-mer not only delivered a suboptimal signal, which blocked subsequent responses to OVA, anti-IgG, and anti-kappa, but also competed for binding with OVA. Our results show that fine-tuning of BCR-ligand recognition can lead to B cell nonresponsiveness, activation, or inhibition

Abstract A24: ZAP-70 and SYK regulation in the B cell receptor pathway in chronic lymphocytic leukemia

Abstract Recent discoveries of inhibitors for Syk (spleen tyrosine kinase) family protein kinases provide promising therapeutic options for patients with chronic lymphocytic leukemia (CLL). However, molecular mechanisms of these inhibitors in the B cell receptor (BCR) signaling pathway are not well understood. This study investigates functionality of Syk family protein kinases and their inhibitors in the BCR signaling pathway in CLL cells. In this work, we tested several computational models of ZAP-70 (ζ chain–associated protein kinase of 70 kD) and SYK regulation in the BCR signaling pathway to explain the observed differences in the clinical behaviors of ZAP-70+ / ZAP-70- phenotypes of CLL patients. Specifically, we characterized the effects of different ZAP-70 and SYK expression and phosphorylation levels on the BCR activation threshold. Using stochastic simulations of BCR signaling network, we reproduced the correlations between the observed and calculated trends quantitatively. We find that an increased ZAP-70 expression is correlated with decreased levels of phosphorylated ZAP-70 and SYK, depending on the amount of SYK that is expressed in cells. Our calculations also show that ZAP-70 is able to compensate for a missing SYK functionality with an increased BCR activation threshold in SYK-deficient B-CLL cells, similar to SYK-deficient B cells. Furthermore, our computational model predicts a dual inhibition of ZAP-70 and SYK for the treatment of ZAP-70+ patients, as a selective inhibition of either ZAP-70 or SYK results in disease relapse. Our findings uncover molecular mechanisms of ZAP-70 and SYK regulation in the BCR signaling pathway for the development of CLL immunotherapies. Citation Format: Maria P. Frushicheva. ZAP-70 and SYK regulation in the B cell receptor pathway in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A24.</jats:p

Abstract B131: Quantitative modeling of Syk regulation in B cell chronic lymphocytic leukemia

Abstract Spleen tyrosine kinases (Syk) are initiators of B cell receptor-mediated (BCR) signaling events, enabling cells to drive chronic lymphocytic leukemia (CLL) progression. However, the mechanistic principles of biochemical signaling pathways that contribute to CLL pathogenesis are poorly understood. This study investigates Syk-mediated molecular pathways of the BCR signaling network using computational modeling. An expression of ζ chain-associated protein kinase of 70 kD (ZAP-70, Syk-family protein kinase) in the BCR signaling pathway is an adverse prognostic marker in CLL. Therefore, we need a better understanding of molecular mechanisms of Syk family protein kinases and effects of their inhibitors in the BCR signaling pathway. In this work, we dissected several computational models of ZAP-70 and SYK regulation to design an integrated mechanistic description of the BCR signaling pathway, and to explain the observed differences in the clinical behaviors of ZAP-70+ / ZAP-70- phenotypes of CLL patients. Specifically, we characterized the effects of different ZAP-70 and SYK expression and phosphorylation levels on the BCR activation threshold, using stochastic simulations of the BCR signaling network. Preliminary findings suggest that ZAP-70 serves as an adaptor protein that facilitates the recruitment of SYK to the BCR complex. We find that the expression of ZAP-70 enhances the BCR signaling, and therefore correlates with a more aggressive disease course. Our results will help in the development of novel therapies targeting Syk functions to regulate B-CLL responses. Citation Format: Maria P. Frushicheva. Quantitative modeling of Syk regulation in B cell chronic lymphocytic leukemia [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B131.</jats:p