scholarly journals Reprogramming protein kinase substrate specificity through synthetic mutations

2016 ◽  
Author(s):  
Joshua M. Lubner ◽  
George M. Church ◽  
Michael F. Chou ◽  
Daniel Schwartz
Keyword(s):  
Protein Kinase ◽  
Substrate Specificity ◽  
Structure Function ◽  
Binding Pocket ◽  
Missense Mutations ◽  
New Paradigm ◽  
Substrate Binding Pocket ◽  
Kinase Substrate ◽  
Substrate Preferences ◽  
Kinase Specificity

Protein kinase specificity is largely imparted through substrate binding pocket motifs. Missense mutations in these regions are frequently associated with human disease, and in some cases can alter substrate specificity. However, current efforts at decoding the influence of mutations on substrate specificity have been focused on disease-associated mutations. Here, we adapted the Proteomic Peptide Library (ProPeL) approach for determining kinase specificity to the task of exploring structure-function relationships in kinase specificity by interrogating the effects of synthetic mutation. We established a specificity model for the wild-type DYRK1A kinase with unprecedented resolution. Using existing crystallographic and sequence homology data, we rationally designed mutations that precisely reprogrammed the DYRK1A kinase at the P+1 position to mimic the substrate preferences of a related kinase, CK II. This study illustrates a new synthetic biological approach to reprogram kinase specificity by design, and a powerful new paradigm to investigate structure-function relationships underpinning kinase substrate specificity.

Expanding Substrate Specificity of ω-Transaminase by Rational Remodeling of a Large Substrate-Binding Pocket

2015 ◽  
Vol 357 (12) ◽  
pp. 2712-2720 ◽  
Author(s):  
Sang-Woo Han ◽  
Eul-Soo Park ◽  
Joo-Young Dong ◽  
Jong-Shik Shin
Keyword(s):  
Substrate Specificity ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket

scholarly journals Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP

2005 ◽  
Vol 389 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Melinda DEMENDI ◽  
Noboru ISHIYAMA ◽  
Joseph S. LAM ◽  
Albert M. BERGHUIS ◽  
Carole CREUZENET
Keyword(s):  
Enzyme Activity ◽  
Substrate Specificity ◽  
Molecular Basis ◽  
Substrate Binding ◽  
Structural Data ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Expected Effect ◽  
Biochemical Assays ◽  
Golden Opportunity

WbpP is the only genuine UDP-GlcNAc (UDP-N-acetylglucosamine) C4 epimerase for which both biochemical and structural data are available. This represents a golden opportunity to elucidate the molecular basis for its specificity for N-acetylated substrates. Based on the comparison of the substrate binding site of WbpP with that of other C4 epimerases that convert preferentially non-acetylated substrates, or that are able to convert both acetylated and non-acetylated substrates equally well, specific residues of WbpP were mutated, and the substrate specificity of the mutants was determined by direct biochemical assays and kinetic analyses. Most of the mutations tested were anticipated to trigger a significant switch in substrate specificity, mostly towards a preference for non-acetylated substrates. However, only one of the mutations (A209H) had the expected effect, and most others resulted in enhanced specificity of WbpP for N-acetylated substrates (Q201E, G102K, Q201E/G102K, A209N and S143A). One mutation (S144K) totally abolished enzyme activity. These data indicate that, although all residues targeted in the present study turned out to be important for catalysis, determinants of substrate specificity are not confined to the substrate-binding pocket and that longer range interactions are essential in allowing proper positioning of various ligands in the binding pocket. Hence prediction or engineering of substrate specificity solely based on sequence analysis, or even on modelling of the binding pocket, might lead to incorrect functional assignments.

scholarly journals The design of peptide-based substrates for the cdc2 protein kinase

1995 ◽  
Vol 309 (3) ◽  
pp. 927-931 ◽  
Author(s):  
J Srinivasan ◽  
M Koszelak ◽  
M Mendelow ◽  
Y G Kwon ◽  
D S Lawrence
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Amino Acid Sequence ◽  
Substrate Specificity ◽  
Bovine Brain ◽  
Sequence Specificity ◽  
Cyclin Dependent Kinase ◽  
Cdc2 Kinase ◽  
Kinase Substrate ◽  
Pisaster Ochraceus

The substrate sequence specificity of the cdc2 protein kinase from Pisaster ochraceus has been evaluated. The peptide, Ac-Ser-Pro-Gly-Arg-Arg-Arg-Arg-Lys-amide, serves as an efficient cdc2 kinase substrate with a Km of 1.50 +/- 0.04 microM and a Vmax. of 12.00 +/- 0.18 mumol/min per mg. The amino acid sequence of this peptide is not based on any sequence in a known protein substrate of the cyclin-dependent kinase, but rather was designed from structural attributes that appear to be important in the majority of cdc2 substrates. The cyclin-dependent enzyme is remarkably indiscriminate in its ability to recognize and phosphorylate peptides that contain an assortment of structurally diverse residues at the P-2, P-1 and P+2 positions. However, peptides that contain a free N-terminal serine or lack an arginine at the P+4 position are relatively poor substrates. These aspects of the substrate specificity of the cdc2 protein kinase are compared and contrasted with the previously reported substrate specificity of a cdc2-like protein kinase from bovine brain [Beaudette, Lew and Wang (1993) J. Biol. Chem. 268, 20825-20830].

Structural basis for substrate specificity of heteromeric transporters of neutral amino acids

2021 ◽  
Vol 118 (49) ◽  
pp. e2113573118
Author(s):  
Carlos F. Rodriguez ◽  
Paloma Escudero-Bravo ◽  
Lucía Díaz ◽  
Paola Bartoccioni ◽  
Carmen García-Martín ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Molecular Mechanisms ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Neutral Amino Acid ◽  
Substrate Preference ◽  
Structural Basis ◽  
Substrate Binding Pocket

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo–electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.

scholarly journals Characterizing Protein Kinase Substrate Specificity Using the Proteomic Peptide Library (ProPeL) Approach

2018 ◽  
Vol 10 (2) ◽  
pp. e38 ◽  
Author(s):  
Joshua M. Lubner ◽  
Jeremy L. Balsbaugh ◽  
George M. Church ◽  
Michael F. Chou ◽  
Daniel Schwartz
Keyword(s):  
Protein Kinase ◽  
Substrate Specificity ◽  
Peptide Library ◽  
Kinase Substrate

scholarly journals Structural Insights for Core Scaffold and Substrate Specificity of B1, B2, and B3 Metallo-β-Lactamases

2022 ◽  
Vol 12 ◽  
Author(s):  
Yeongjin Yun ◽  
Sangjun Han ◽  
Yoon Sik Park ◽  
Hyunjae Park ◽  
Dogyeong Kim ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Chemical Structure ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Structural Features ◽  
Amino Acid Sequences ◽  
Zinc Ions ◽  
Substrate Binding Pocket ◽  
Inhibitory Spectrum

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems; however, no effective inhibitors are currently clinically available. MBLs are classified into three subclasses: B1, B2, and B3. Although the amino acid sequences of MBLs are varied, their overall scaffold is well conserved. In this study, we systematically studied the primary sequences and crystal structures of all subclasses of MBLs, especially the core scaffold, the zinc-coordinating residues in the active site, and the substrate-binding pocket. We presented the conserved structural features of MBLs in the same subclass and the characteristics of MBLs of each subclass. The catalytic zinc ions are bound with four loops from the two central β-sheets in the conserved αβ/βα sandwich fold of MBLs. The three external loops cover the zinc site(s) from the outside and simultaneously form a substrate-binding pocket. In the overall structure, B1 and B2 MBLs are more closely related to each other than they are to B3 MBLs. However, B1 and B3 MBLs have two zinc ions in the active site, while B2 MBLs have one. The substrate-binding pocket is different among all three subclasses, which is especially important for substrate specificity and drug resistance. Thus far, various classes of β-lactam antibiotics have been developed to have modified ring structures and substituted R groups. Currently available structures of β-lactam-bound MBLs show that the binding of β-lactams is well conserved according to the overall chemical structure in the substrate-binding pocket. Besides β-lactam substrates, B1 and cross-class MBL inhibitors also have distinguished differences in the chemical structure, which fit well to the substrate-binding pocket of MBLs within their inhibitory spectrum. The systematic structural comparison among B1, B2, and B3 MBLs provides in-depth insight into their substrate specificity, which will be useful for developing a clinical inhibitor targeting MBLs.

Sign in / Sign up

Export Citation Format

Share Document