Carolyn J. Adamski

Deep Sequencing of Systematic Combinatorial Libraries Reveals β-Lactamase Sequence Constraints at High Resolution

In this study, combinatorial libraries were used in conjunction with ultrahigh-throughput sequencing to comprehensively determine the impact of each of the 19 possible amino acid substitutions at each residue position in the TEM-1 β-lactamase enzyme. The libraries were introduced into Escherichiacoli, and mutants were selected for ampicillin resistance. The selected colonies were pooled and subjected to ultrahigh-throughput sequencing to reveal the sequence preferences at each position. The depth of sequencing provided a clear, statistically significant picture of what amino acids are favored for ampicillin hydrolysis for all 263 positions of the enzyme in one experiment. Although the enzyme is generally tolerant of amino acid substitutions, several surface positions far from the active site are sensitive to substitutions suggesting a role for these residues in enzyme stability, solubility, or catalysis. In addition, information on the frequency of substitutions was used to identify mutations that increase enzyme thermodynamic stability. Finally, a comparison of sequence requirements based on the mutagenesis results versus those inferred from sequence conservation in an alignment of 156 class A β-lactamases reveals significant differences in that several residues in TEM-1 do not tolerate substitutions and yet extensive variation is observed in the alignment and vice versa. An analysis of the TEM-1 and other class A structures suggests that residues that vary in the alignment may nevertheless make unique, but important, interactions within individual enzymes.

Molecular basis for the catalytic specificity of the CTX-M extended-spectrum β-lactamases.

Extended-spectrum β-lactamases (ESBLs) pose a threat to public health because of their ability to confer resistance to extended-spectrum cephalosporins such as cefotaxime. The CTX-M β-lactamases are the most widespread ESBL enzymes among antibiotic resistant bacteria. Many of the active site residues are conserved between the CTX-M family and non-ESBL β-lactamases such as TEM-1, but the residues Ser237 and Arg276 are specific to the CTX-M family, suggesting that they may help to define the increased specificity for cefotaxime hydrolysis. To test this hypothesis, site-directed mutagenesis of these positions was performed in the CTX-M-14 β-lactamase. Substitutions of Ser237 and Arg276 with their TEM-1 counterparts, Ala237 and Asn276, had a modest effect on cefotaxime hydrolysis, as did removal of the Arg276 side chain in an R276A mutant. The S237A:R276N and S237A:R276A double mutants, however, exhibited 29- and 14-fold losses in catalytic efficiency for cefotaxime hydrolysis, respectively, while the catalytic efficiency for benzylpenicillin hydrolysis was unchanged. Therefore, together, the Ser237 and Arg276 residues are important contributors to the cefotaximase substrate profile of the enzyme. High-resolution crystal structures of the CTX-M-14 S70G, S70G:S237A, and S70G:S237A:R276A variants alone and in complex with cefotaxime show that residues Ser237 and Arg276 in the wild-type enzyme promote the expansion of the active site to accommodate cefotaxime and favor a conformation of cefotaxime that allows optimal contacts between the enzyme and substrate. The conservation of these residues, linked to their effects on structure and catalysis, imply that their coevolution is an important specificity determinant in the CTX-M family.

Inhibition of ERK1/2 Restores GSK3β Activity and Protein Synthesis Levels in a Model of Tuberous Sclerosis

Tuberous sclerosis (TS) is a multi-organ autosomal dominant disorder that is best characterized by neurodevelopmental deficits and the presence of benign tumors. TS pathology is caused by mutations in tuberous sclerosis complex (TSC) genes and is associated with insulin resistance, decreased glycogen synthase kinase 3β (GSK3β) activity, activation of the mammalian target of rapamycin complex 1 (mTORC1), and subsequent increase in protein synthesis. Here, we show that extracellular signal–regulated kinases (ERK1/2) respond to insulin stimulation and integrate insulin signaling to phosphorylate and thus inactivate GSK3β, resulting in increased protein synthesis that is independent of Akt/mTORC1 activity. Inhibition of ERK1/2 in Tsc2−/− cells—a model of TS—rescues GSK3β activity and protein synthesis levels, thus highlighting ERK1/2 as a potential therapeutic target for the treatment of TS.

Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine β-Lactamases by Relieving Steric Strain

Serine β-lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. They utilize an active-site serine residue as a nucleophile, forming an acyl-enzyme intermediate during hydrolysis. In this study, thermal denaturation experiments as well as X-ray crystallography were performed to test the effect of substitution of the catalytic serine with glycine on protein stability in serine β-lactamases. Six different enzymes comprising representatives from each of the three classes of serine β-lactamases were examined, including TEM-1, CTX-M-14, and KPC-2 of class A, P99 of class C, and OXA-48 and OXA-163 of class D. For each enzyme, the wild type and a serine-to-glycine mutant were evaluated for stability. The glycine mutants all exhibited enhanced thermostability compared to that of the wild type. In contrast, alanine substitutions of the catalytic serine in TEM-1, OXA-48, and OXA-163 did not alter stability, suggesting removal of the Cβ atom is key to the stability increase associated with the glycine mutants. The X-ray crystal structures of P99 S64G, OXA-48 S70G and S70A, and OXA-163 S70G suggest that removal of the side chain of the catalytic serine releases steric strain to improve enzyme stability. Additionally, analysis of the torsion angles at the nucleophile position indicates that the glycine mutants exhibit improved distance and angular parameters of the intrahelical hydrogen bond network compared to those of the wild-type enzymes, which is also consistent with increased stability. The increased stability of the mutants indicates that the enzyme pays a price in stability for the presence of a side chain at the catalytic serine position but that the cost is necessary in that removal of the serine drastically impairs function. These findings support the stability-function hypothesis, which states that active-site residues are optimized for substrate binding and catalysis but that the requirements for catalysis are often not consistent with the requirements for optimal stability.

Role of β‐lactamase residues in a common interface for binding the structurally unrelated inhibitory proteins BLIP and BLIP‐II

The β‐lactamase inhibitory proteins (BLIPs) are a model system for examining molecular recognition in protein‐protein interactions. BLIP and BLIP‐II are structurally unrelated proteins that bind and inhibit TEM‐1 β‐lactamase. Both BLIPs share a common binding interface on TEM‐1 and make contacts with many of the same TEM‐1 surface residues. BLIP‐II, however, binds TEM‐1 over 150‐fold tighter than BLIP despite the fact that it has fewer contact residues and a smaller binding interface. The role of eleven TEM‐1 amino acid residues that contact both BLIP and BLIP‐II was examined by alanine mutagenesis and determination of the association (kon) and dissociation (koff) rate constants for binding each partner. The substitutions had little impact on association rates and resulted in a wide range of dissociation rates as previously observed for substitutions on the BLIP side of the interface. The substitutions also had less effect on binding affinity for BLIP than BLIP‐II. This is consistent with the high affinity and small binding interface of the TEM‐1‐BLIP‐II complex, which predicts per residue contributions should be higher for TEM‐1 binding to BLIP‐II versus BLIP. Two TEM‐1 residues (E104 and M129) were found to be hotspots for binding BLIP while five (L102, Y105, P107, K111, and M129) are hotspots for binding BLIP‐II with only M129 as a common hotspot for both. Thus, although the same TEM‐1 surface binds to both BLIP and BLIP‐II, the distribution of binding energy on the surface is different for the two target proteins, that is, different binding strategies are employed.

Systematic substitutions at BLIP position 50 result in changes in binding specificity for class A β-lactamases

BackgroundThe production of β-lactamases by bacteria is the most common mechanism of resistance to the widely prescribed β-lactam antibiotics. β-lactamase inhibitory protein (BLIP) competitively inhibits class A β-lactamases via two binding loops that occlude the active site. It has been shown that BLIP Tyr50 is a specificity determinant in that substitutions at this position result in large differential changes in the relative affinity of BLIP for class A β-lactamases.ResultsIn this study, the effect of systematic substitutions at BLIP position 50 on binding to class A β-lactamases was examined to further explore the role of BLIP Tyr50 in modulating specificity. The results indicate the sequence requirements at position 50 are widely different depending on the target β-lactamase. Stringent sequence requirements were observed at Tyr50 for binding Bacillus anthracis Bla1 while moderate requirements for binding TEM-1 and relaxed requirements for binding KPC-2 β-lactamase were seen. These findings cannot be easily rationalized based on the β-lactamase residues in direct contact with BLIP Tyr50 since they are identical for Bla1 and KPC-2 suggesting that differences in the BLIP-β-lactamase interface outside the local environment of Tyr50 influence the effect of substitutions.ConclusionsResults from this study and previous studies suggest that substitutions at BLIP Tyr50 may induce changes at the interface outside its local environment and point to the complexity of predicting the impact of substitutions at a protein-protein interaction interface.

Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations.

CphA is a Zn2+-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function.

BLIP-II Employs Differential Hotspot Residues To Bind Structurally Similar Staphylococcus aureus PBP2a and Class A β-Lactamases

The interaction of β-lactamase inhibitory protein II (BLIP-II) with β-lactamases serves as a model system to investigate the principles underlying protein-protein interactions. Previous studies have focused on identifying the determinants of binding affinity and specificity between BLIP-II and class A β-lactamases. However, interactions between BLIP-II and other bacterial proteins have yet to be explored. Here, we provide evidence that BLIP-II binds penicillin binding protein 2a (PBP2a) from methicillin-resistant Staphylococcus aureus (MRSA) with a KD in the low micromolar range. In comparison to the binding constants for the potent interaction between BLIP-II and TEM-1 β-lactamase (KD = 0.5 pM), the on-rate for BLIP-II binding PBP2a is 44 000 times slower and the off-rate is 170 times faster. Therefore, a slow association rate is a limiting factor for the potency of the interaction between BLIP-II and PBP2a. Results from alanine scanning mutagenesis of the predicted interface residues of BLIP-II indicate that charged residues on the periphery of the BLIP-II interface play a critical role for binding PBP2a, in contrast to previous findings that aromatic residues at the center of the BLIP-II interface are critical for the interaction with β-lactamases. Interestingly, many of the alanine mutants at the BLIP-II interface increase kon for binding PBP2a, consistent with the association rate being a limiting factor for affinity. In summary, the results of the study reveal that BLIP-II binds PBP2a, although weakly compared to binding of β-lactamases, and provides insights into the different binding strategies used for these targets.

Reduction of protein kinase A-mediated phosphorylation of ATXN1-S776 in Purkinje cells delays onset of Ataxia in a SCA1 mouse model

Spinocerebellar ataxia type 1 (SCA1) is a polyglutamine (polyQ) repeat neurodegenerative disease in which a primary site of pathogenesis are cerebellar Purkinje cells. In addition to polyQ expansion of ataxin-1 protein (ATXN1), phosphorylation of ATXN1 at the serine 776 residue (ATXN1-pS776) plays a significant role in protein toxicity. Utilizing a biochemical approach, pharmacological agents and cell-based assays, including SCA1 patient iPSC-derived neurons, we examine the role of Protein Kinase A (PKA) as an effector of ATXN1-S776 phosphorylation. We further examine the implications of PKA-mediated phosphorylation at ATXN1-S776 on SCA1 through genetic manipulation of the PKA catalytic subunit Cα in Pcp2-ATXN1[82Q] mice. Here we show that pharmacologic inhibition of S776 phosphorylation in transfected cells and SCA1 patient iPSC-derived neuronal cells lead to a decrease in ATXN1. In vivo, reduction of PKA-mediated ATXN1-pS776 results in enhanced degradation of ATXN1 and improved cerebellar-dependent motor performance. These results provide evidence that PKA is a biologically important kinase for ATXN1-pS776 in cerebellar Purkinje cells.

CLN8 is an endoplasmic reticulum cargo receptor that regulates lysosome biogenesis

Organelle biogenesis requires proper transport of proteins from their site of synthesis to their target subcellular compartment1–3. Lysosomal enzymes are synthesized in the endoplasmic reticulum (ER) and traffic through the Golgi complex before being transferred to the endolysosomal system4–6, but how they are transferred from the ER to the Golgi is unknown. Here, we show that ER-to-Golgi transfer of lysosomal enzymes requires CLN8, an ER-associated membrane protein whose loss of function leads to the lysosomal storage disorder, neuronal ceroid lipofuscinosis 8 (a type of Batten disease)7. ER-to-Golgi trafficking of CLN8 requires interaction with the COPII and COPI machineries via specific export and retrieval signals localized in the cytosolic carboxy terminus of CLN8. CLN8 deficiency leads to depletion of soluble enzymes in the lysosome, thus impairing lysosome biogenesis. Binding to lysosomal enzymes requires the second luminal loop of CLN8 and is abolished by some disease-causing mutations within this region. Our data establish an unanticipated example of an ER receptor serving the biogenesis of an organelle and indicate that impaired transport of lysosomal enzymes underlies Batten disease caused by mutations in CLN8.di Ronza et al. identify CLN8 as a cargo receptor for lysosomal enzymes required for their endoplasmic-reticulum-to-Golgi transport, linking Batten disease caused by CLN8 mutations to defects in organelle biogenesis.