Wolfgang Peti

Structures of Dynamic Protein Complexes: Hybrid Techniques to Study MAP Kinase Complexes and the ESCRT System

The integration of complementary molecular methods (including X-ray crystallography, NMR spectroscopy, small angle X-ray/neutron scattering, and computational techniques) is frequently required to obtain a comprehensive understanding of dynamic macromolecular complexes. In particular, these techniques are critical for studying intrinsically disordered protein regions (IDRs) or intrinsically disordered proteins (IDPs) that are part of large protein:protein complexes. Here, we explain how to prepare IDP samples suitable for study using NMR spectroscopy, and describe a novel SAXS modeling method (ensemble refinement of SAXS; EROS) that integrates the results from complementary methods, including crystal structures and NMR chemical shift perturbations, among others, to accurately model SAXS data and describe ensemble structures of dynamic macromolecular complexes.

KNL1 Binding to PP1 and Microtubules Is Mutually Exclusive

The kinetochore scaffold 1 (KNL1) protein coordinates the spindle assembly checkpoint (SAC), a signaling pathway that delays chromosome segregation until all sister chromatids are properly attached to spindle microtubules. Recently, microtubules and protein phosphatase 1 (PP1), which both bind the N-terminal domain of KNL1, have emerged as regulators of the SAC; however, how these proteins interact to contribute to SAC signaling is unknown. Here, we use X-ray crystallography, nuclear magnetic resonance spectroscopy, and biochemical assays to show how KNL1 binds both PP1 and microtubules. Unexpectedly, we discovered that PP1 and microtubules bind KNL1 via overlapping binding sites. Further, we showed that Aurora B kinase phosphorylation results in distinct patterns of KNL1 complex disruption. Finally, combining this data with co-sedimentation assays unequivocally demonstrated that microtubules and PP1 binding to KNL1 is mutually exclusive, with preferential formation of the KNL1:PP1 holoenzyme in the presence of PP1.

Dynamic activation and regulation of the mitogen-activated protein kinase p38

Significance The physical basis for the regulation of protein MAP kinases is key to understanding their role in cellular signaling in human biology and disease. However, the biochemical events that lead to MAPK activation are not well understood. Here, using biomolecular NMR dynamics measurements, we show that kinase activation is not due simply to phosphorylation of the activation loop. Rather, we show that phosphorylation and substrate binding cause the dynamics of p38 to change from a state in which they are uncoordinated to one that is uniform across the entire molecule. That is, synchronized dynamics coordinates enzyme activity, which leads to a shift in the energy landscape and ultimately drives enzyme function. This makes essential elements of allostery and activation dynamically accessible. Mitogen-activated protein kinases, which include p38, are essential for cell differentiation and autophagy. The current model for p38 activation involves activation-loop phosphorylation with subsequent substrate binding leading to substrate phosphorylation. Despite extensive efforts, the molecular mechanism of activation remains unclear. Here, using NMR spectroscopy, we show how the modulation of protein dynamics across timescales activates p38. We find that activation-loop phosphorylation does not change the average conformation of p38; rather it quenches the loop ps-ns dynamics. We then show that substrate binding to nonphosphorylated and phosphorylated p38 results in uniform µs-ms backbone dynamics at catalytically essential regions and across the entire molecule, respectively. Together, these results show that phosphorylation and substrate binding flatten the energy landscape of the protein, making essential elements of allostery and activation dynamically accessible. The high degree of structural conservation among ser/thr kinases suggests that elements of this mechanism may be conserved across the kinase family.

Preparation of Phosphorylated Proteins for NMR Spectroscopy

Phosphorylation is a ubiquitous posttranslational modification that is essential for the regulation of many cellular processes. The human genome consists of more than 200,000 phosphorylation sites, whose phosphorylation is tightly controlled by ≥500 kinases and ~200 phosphatases. Given the large number of phosphorylation sites and the key role phosphorylation plays in regulating cellular processes, it is essential to characterize the impact of phosphorylation on substrate structure, dynamics, and function. However, a major challenge is the large-scale production of phosphorylated proteins in vitro for these structural, functional, and dynamic studies. Here, we describe an efficient protocol used routinely in our laboratory for the production of phosphorylated proteins. We also describe the methods used for identifying, characterizing, and separating the resulting phosphorylated proteins for subsequent studies.

Structural and Regulatory Changes in PBP4 Trigger Decreased β-Lactam Susceptibility in Enterococcus faecalis

ABSTRACT Enterococcus faecalis strains resistant to penicillin and ampicillin are rare and have been associated with increases in quantities of low-affinity penicillin-binding protein 4 (PBP4) or with amino acid substitutions in PBP4. We report an E. faecalis strain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yielded E. faecalis with MICs of penicillins and carbapenems higher than those for wild-type strain E. faecalis JH2-2. Sequence analysis of the pbp4 gene of LS4828 compared to that of JH2-2 revealed two point mutations with amino acid substitutions (V223I, A617T) and deletion of an adenine from the region upstream of the predicted pbp4 −35 promoter sequence (UP region). Purified PBP4 from LS4828 exhibited less affinity for Bocillin FL than did PBP4 from JH2-2, which was recapitulated by purified PBP4 containing only the A617T mutation. Differential scanning fluorimetry studies showed that the LS4828 and A617T variants are destabilized compared to wild-type PBP4. Further, reverse transcription-PCR indicated increased transcription of pbp4 in LS4828 and Western blot analysis with polyclonal PBP4 antibody revealed greater quantities of PBP4 in LS4828 than in JH2-2 lysates and membrane preparations. Placing the promoter regions from LS4828 or JH2-2 upstream of a green fluorescent protein reporter gene confirmed that the adenine deletion was associated with increased transcription. Together, these data suggest that the reduced susceptibility to β-lactam antibiotics observed in E. faecalis LS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems. IMPORTANCE Enterococcus faecalis is an important cause of community-acquired and nosocomial infections and creates therapeutic dilemmas because of its frequent resistance to several classes of antibiotics. We report an E. faecalis strain with decreased ampicillin and imipenem susceptibility isolated after prolonged courses of aminopenicillin therapy for a prosthetic joint infection. Its reduced susceptibility is attributable to a combination of increased quantities of low-affinity PBP4 and an amino acid substitution in proximity to the active site that destabilizes the protein. Our findings provide a cautionary tale for clinicians who elect to “suppress” infections in prosthetic joints and offer novel insights into the interaction of β-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains. Enterococcus faecalis is an important cause of community-acquired and nosocomial infections and creates therapeutic dilemmas because of its frequent resistance to several classes of antibiotics. We report an E. faecalis strain with decreased ampicillin and imipenem susceptibility isolated after prolonged courses of aminopenicillin therapy for a prosthetic joint infection. Its reduced susceptibility is attributable to a combination of increased quantities of low-affinity PBP4 and an amino acid substitution in proximity to the active site that destabilizes the protein. Our findings provide a cautionary tale for clinicians who elect to “suppress” infections in prosthetic joints and offer novel insights into the interaction of β-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains.

A Sephin1-insensitive tripartite holophosphatase dephosphorylates translation initiation factor 2α.

The integrated stress response (ISR) is regulated by kinases that phosphorylate the α subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2αP-directed holophosphatase, a therapeutic target in diseases of protein misfolding, is comprised of a regulatory subunit, PPP1R15, and a catalytic subunit, protein phosphatase 1 (PP1). In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in the essential function of eIF2αP dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific dephosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the holoenzyme to the [(o-chlorobenzylidene)amino]guanidines Sephin1 or guanabenz, putative small-molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat–containing region in the PPP1R15A regulatory subunit might influence the requirement for G-actin and sensitivity of the holoenzyme to inhibitors. We found that eIF2αP dephosphorylation by PP1 was moderately stimulated by repeat-containing PPP1R15A in an unphysiological low ionic strength buffer, whereas stimulation imparted by the co-presence of PPP1R15A and G-actin was observed under a broad range of conditions, low and physiological ionic strength, regardless of whether the PPP1R15A regulatory subunit had or lacked the N-terminal repeat–containing region and whether it was paired with native PP1 purified from rabbit muscle or recombinant PP1 purified from bacteria. Furthermore, none of the PPP1R15A-containing holophosphatases tested were inhibited by Sephin1 or guanabenz.

Choice of PP1 catalytic subunit affects neither the requirement for G-actin nor the insensitivity to Sephin1 of PPP1R15A-regulated eIF2αP dephosphorylation

The integrated stress response is regulated by kinases that phosphorylate translation initiation factor 2α and phosphatases that dephosphorylate it. Genetic and biochemical data indicate that the eIF2α-directed holophosphatase - a therapeutic target in diseases of protein misfolding - is comprised of a regulatory, PPP1R15, and a catalytic, Protein Phosphatase 1 (PP1), subunit. However, differing reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific de-phosphorylation1-3. An additional concern relates to the sensitivity of this PP1 holoenzyme to the [(o-chlorobenzylidene)amino]guanidines (Sephin1 or Guanabenz) small molecule proteostasis modulators3,4. We find that in the absence of G-actin, PPP1R15A regulatory subunit fragments were unable to accelerate eIF2α dephosphorylation beyond that affected by a catalytic subunit alone, whether PP1 was purified from rabbit muscle or from bacteria. Furthermore, we did not observe Sephin1 or Guanabenz inhibition of eIF2α dephosphorylation by any PPP1R15A-containing holophosphatase.