New surface contacts formed upon reductive lysine methylation: Improving the probability of protein crystallization

Surface lysine methylation (SLM) is a technique for improving the rate of success of protein crystallization by chemically methylating lysine residues. The exact mechanism by which SLM enhances crystallization is still not clear. To study these mechanisms, and to analyze the conditions where SLM will provide the optimal benefits for rescuing failed crystallization experiments, we compared 40 protein structures containing N,N-dimethyl-lysine (dmLys) to a nonredundant set of 18,972 nonmethylated structures from the PDB. By measuring the relative frequency of intermolecular contacts (where contacts are defined as interactions between the residues in proximity with a distance of 3.5 Å or less) of basic residues in the methylated versus nonmethylated sets, dmLys-Glu contacts are seen more frequently than Lys-Glu contacts. Based on observation of the 10 proteins with both native and methylated structures, we propose that the increased rate of contact for dmLys-Glu is due to both a slight increase in the number of amine-carboxyl H-bonds and to the formation of methyl C–H···O interactions. By comparing the relative contact frequencies of dmLys with other residues, the mechanism by which methylation of lysines improves the formation of crystal contacts appears to be similar to that of Lys to Arg mutation. Moreover, analysis of methylated structures with the surface entropy reduction (SER) prediction server suggests that in many cases SLM of predicted SER sites may contribute to improved crystallization. Thus, tools that analyze protein sequences and mark residues for SER mutation may identify proteins with good candidate sites for SLM.     

Toward rational protein crystallization: A Web server for the design of crystallizable protein variants

Growing well-diffracting crystals constitutes a serious bottleneck in structural biology. A recently proposed crystallization methodology for "stubborn crystallizers" is to engineer surface sequence variants designed to form intermolecular contacts that could support a crystal lattice. This approach relies on the concept of surface entropy reduction (SER), i.e., the replacement of clusters of flexible, solvent-exposed residues with residues with lower conformational entropy. This strategy minimizes the loss of conformational entropy upon crystallization and renders crystallization thermodynamically favorable. The method has been successfully used to crystallize more than 15 novel proteins, all stubborn crystallizers. But the choice of suitable sites for mutagenesis is not trivial. Herein, we announce a Web server, the surface entropy reduction prediction server (SERp server), designed to identify mutations that may facilitate crystallization. Suggested mutations are predicted based on an algorithm incorporating a conformational entropy profile, a secondary structure prediction, and sequence conservation. Minor considerations include the nature of flanking residues and gaps between mutation candidates. While designed to be used with default values, the server has many user-controlled parameters allowing for considerable flexibility. Within, we discuss (1) the methodology of the server, (2) how to interpret the results, and (3) factors that must be considered when selecting mutations. We also attempt to benchmark the server by comparing the server's predictions with successful SER structures. In most cases, the structure yielding mutations were easily identified by the SERp server. The server can be accessed at http://www.doe-mbi.ucla.edu/Services/SER.     

Application of the NZ‐1 Fab as a crystallization chaperone for PA tag‐inserted target proteins

An antibody fragment that recognizes the tertiary structure of a target protein with high affinity can be utilized as a crystallization chaperone. Difficulties in establishing conformation‐specific antibodies, however, limit the applicability of antibody fragment‐assisted crystallization. Here, we attempted to establish an alternative method to promote the crystallization of target proteins using an already established anti‐tag antibody. The monoclonal antibody NZ‐1 recognizes the PA tag with an extremely high affinity. It was also established that the PA tag is accommodated in the antigen‐binding pocket in a bent conformation, compatible with an insertion into loop regions on the target. We, therefore, explored the application of NZ‐1 Fab as a crystallization chaperone that complexes with a target protein displaying a PA tag. Specifically, we inserted the PA tag into the β‐hairpins of the PDZ tandem fragment of a bacterial Site‐2 protease. We crystallized the PA‐inserted PDZ tandem mutants with the NZ‐1 Fab and solved the co‐crystal structure to analyze their interaction modes. Although the initial insertion designs produced only moderate‐resolution structures, eliminating the solvent‐accessible space between the NZ‐1 Fab and target PDZ tandem improved the diffraction qualities remarkably. Our results demonstrate that the NZ‐1‐PA system efficiently promotes crystallization of the target protein. The present work also suggests that β‐hairpins are suitable sites for the PA insertion because the PA tag contains a Pro‐Gly sequence with a propensity for a β‐turn conformation.     

The modules of trans‐acyltransferase assembly lines redefined with a central acyl carrier protein

Here, the term “module” is redefined for trans‐acyltransferase (trans‐AT) assembly lines to agree with how its domains cooperate and evolutionarily co‐migrate. The key domain in both the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) modules of assembly lines is the acyl carrier protein (ACP). ACPs not only relay growing acyl chains through the assembly line but also collaborate with enzymes in modules, both in cis and in trans, to add a specific chemical moiety. A ketosynthase (KS) downstream of ACP often plays the role of gatekeeper, ensuring that only a single intermediate generated by the enzymes of a module is passed downstream. Bioinformatic analysis of 526 ACPs from 33 characterized trans‐AT assembly lines reveals ACPs from the same module type generally clade together, reflective of the co‐evolution of these domains with their cognate enzymes. While KSs downstream of ACPs from the same module type generally also clade together, KSs upstream of ACPs do not—in disagreement with the traditional definition of a module. Beyond nomenclature, the presented analysis impacts our understanding of module function, the evolution of assembly lines, pathway prediction, and assembly line engineering.     

Identification of B‐cell epitopes of Borrelia burgdorferi outer surface protein C by screening a phage‐displayed gene fragment library

Outer surface protein C (OspC) of Borrelia stimulates remarkable immune responses during early infection and is therefore currently considered a leading diagnostic and vaccine candidate. The sensitivity and specificity of serological tests based on whole protein OspC for diagnosis of Lyme disease are still unsatisfactory. Minimal B‐cell epitopes are key in the development of reliable immunodiagnostic tools. Using OspC fragments displayed on phage particles (phage library) and anti‐OspC antibodies isolated from sera of naturally infected patients, six OspC epitopes capable of distinguishing between LD patient and healthy control sera were identified. Three of these epitopes are located at the N‐terminus (OspC E1 aa19–27, OspC E2 aa38–53, OspC E3 aa62–66) and three at the C‐terminal end (OspC E4 aa155–163, OspC E5 aa184–190 and OspC E6 aa201–207). OspC E1, E4 and E6 were highly conserved among LD related Borreliae. To our knowledge, epitopes OspC E2, E3 and E5 were identified for the first time in this study. Minimal B‐cell epitopes may provide fundamental data for the development of multi‐epitope‐based diagnostic tools for Lyme disease.     

A novel signal transduction protein: Combination of solute binding and tandem PAS‐like sensor domains in one polypeptide chain

We report the structural and biochemical characterization of a novel periplasmic ligand‐binding protein, Dret_0059, from Desulfohalobium retbaense DSM 5692, an organism isolated from Lake Retba, in Senegal. The structure of the protein consists of a unique combination of a periplasmic solute binding protein (SBP) domain at the N‐terminal and a tandem PAS‐like sensor domain at the C‐terminal region. SBP domains are found ubiquitously, and their best known function is in solute transport across membranes. PAS‐like sensor domains are commonly found in signal transduction proteins. These domains are widely observed as parts of many protein architectures and complexes but have not been observed previously within the same polypeptide chain. In the structure of Dret_0059, a ketoleucine moiety is bound to the SBP, whereas a cytosine molecule is bound in the distal PAS‐like domain of the tandem PAS‐like domain. Differential scanning flourimetry support the binding of ligands observed in the crystal structure. There is significant interaction between the SBP and tandem PAS‐like domains, and it is possible that the binding of one ligand could have an effect on the binding of the other. We uncovered three other proteins with this structural architecture in the non‐redundant sequence data base, and predict that they too bind the same substrates. The genomic context of this protein did not offer any clues for its function. We did not find any biological process in which the two observed ligands are coupled. The protein Dret_0059 could be involved in either signal transduction or solute transport.     

Conformational changes within the HLA‐A1:MAGE‐A1 complex induced by binding of a recombinant antibody fragment with TCR‐like specificity

Although there is X‐ray crystallographic evidence that the interaction between major histocompatibility complex (MHC, in humans HLA) class I molecules and T cell receptors (TCR) or killer cell Ig‐like receptors (KIR) may be accompanied by considerable changes in the conformation of selected residues or even entire loops within TCR or KIR, conformational changes between receptor‐bound and ‐unbound MHC class I molecules of comparable magnitude have not been observed so far. We have previously determined the structure of the MHC class I molecule HLA‐A1 bound to a melanoma antigen‐encoding gene (MAGE)‐A1‐derived peptide in complex with a recombinant antibody fragment with TCR‐like specificity, Fab‐Hyb3. Here, we compare the X‐ray structure of HLA‐A1:MAGE‐A1 with that complexed with Fab‐Hyb3 to gain insight into structural changes of the MHC molecule that might be induced by the interaction with the antibody fragment. Apart from the expulsion of several water molecules from the interface, Fab‐Hyb3 binding results in major rearrangements (up to 5.5 Å) of heavy chain residues Arg65, Gln72, Arg145, and Lys146. Residue 65 is frequently and residues 72 and 146 are occasionally involved in TCR binding‐induced conformational changes, as revealed by a comparison with MHC class I structures in TCR‐liganded and ‐unliganded forms. On the other hand, residue 145 is subject to a reorientation following engagement of HLA‐Cw4 and KIR2DL1. Therefore, conformational changes within the HLA‐A1:MAGE‐A1:Fab‐Hyb3 complex include MHC residues that are also involved in reorientations in complexes with natural ligands, pointing to their central importance for the peptide‐dependent recognition of MHC molecules.     

MQAPsingle: A quasi single‐model approach for estimation of the quality of individual protein structure models

We present a Model Quality Assessment Program (MQAP), called MQAPsingle, for ranking and assessing the absolute global quality of single protein models. MQAPsingle is quasi single‐model MQAP, a method that combines advantages of both “pure” single‐model MQAPs and clustering MQAPs. This approach results in higher accuracy compared to the state‐of‐the‐art single‐model MQAPs. Notably, the prediction for a given model is the same regardless if this model is submitted to our server alone or together with other models. Proteins 2016; 84:1021–1028. © 2015 Wiley Periodicals, Inc.     

'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.     

Docking and molecular dynamics simulations of the Fyn‐SH3 domain with free and phospholipid bilayer‐associated 18.5‐kDa myelin basic protein (MBP)—Insights into a noncanonical and fuzzy interaction

The molecular details of the association between the human Fyn‐SH3 domain, and the fragment of 18.5‐kDa myelin basic protein (MBP) spanning residues S38–S107 (denoted as xα2‐peptide, murine sequence numbering), were studied in silico via docking and molecular dynamics over 50‐ns trajectories. The results show that interaction between the two proteins is energetically favorable and heavily dependent on the MBP proline‐rich region (P93‐P98) in both aqueous and membrane environments. In aqueous conditions, the xα2‐peptide/Fyn‐SH3 complex adopts a “sandwich”‐like structure. In the membrane context, the xα2‐peptide interacts with the Fyn‐SH3 domain via the proline‐rich region and the β‐sheets of Fyn‐SH3, with the latter wrapping around the proline‐rich region in a form of a clip. Moreover, the simulations corroborate prior experimental evidence of the importance of upstream segments beyond the canonical SH3‐ligand. This study thus provides a more‐detailed glimpse into the context‐dependent interaction dynamics and importance of the β‐sheets in Fyn‐SH3 and proline‐rich region of MBP. Proteins 2017; 85:1336–1350. © 2017 Wiley Periodicals, Inc.     

Improved protective efficacy of a chimeric Staphylococcus aureus vaccine candidate iron‐regulated surface determinant B (N126–P361)‐target of RNAIII activating protein in mice

The pathogen Staphylococcus aureus causes a wide range of serious infections, necessitating urgent development of a vaccine against this organism. However, currently developed vaccines are relatively ineffective because of the limited antigenic component that is contained in the vaccine formulations. To develop an effective S. aureus candidate vaccine, overlapping PCR was used to add the truncated immunodominant antigen iron‐regulated surface determinant B (IsdB)_{(N126–P361)} (tIsdB) to the N‐terminal of intact antigen target of RNAIII activating protein (TRAP) and thus construct a tIsdB‐TRAP chimera. The humoral and cellular immune responses against tIsdB‐TRAP were compared with those against single or combined formulations. tIsdB‐TRAP elicited significantly stronger humoral responses in mice (P < 0.05). As to cellular immune responses in mice, the tIsdB‐TRAP group resulted in a greater IL‐4 response than did other groups (P < 0.05). Greater amounts of IL‐2 and IFN‐γ were found in the tIsdB‐TRAP group. Mouse challenge also showed that tIsdB‐TRAP provided better protection against S. aureus than did the control groups. These results suggest that this chimeric protein may be a promising pathogen target for further vaccine development.     

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X‐ray crystallography has been a powerful approach to understand protein‐protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX‐MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX‐MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX‐MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ‐Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein‐protein complexes, and is an excellent strategy to optimize constructs for high‐resolution structural approaches.     

TSAR,a new graph–theoretical approach to computational modeling of protein side‐chain flexibility: Modeling of ionization properties of proteins

A new graph–theoretical approach called thermodynamic sampling of amino acid residues (TSAR) has been elaborated to explicitly account for the protein side chain flexibility in modeling conformation‐dependent protein properties. In TSAR, a protein is viewed as a graph whose nodes correspond to structurally independent groups and whose edges connect the interacting groups. Each node has its set of states describing conformation and ionization of the group, and each edge is assigned an array of pairwise interaction potentials between the adjacent groups. By treating the obtained graph as a belief‐network—a well‐established mathematical abstraction—the partition function of each node is found. In the current work we used TSAR to calculate partition functions of the ionized forms of protein residues. A simplified version of a semi‐empirical molecular mechanical scoring function, borrowed from our Lead Finder docking software, was used for energy calculations. The accuracy of the resulting model was validated on a set of 486 experimentally determined pK_a values of protein residues. The average correlation coefficient (R) between calculated and experimental pK_a values was 0.80, ranging from 0.95 (for Tyr) to 0.61 (for Lys). It appeared that the hydrogen bond interactions and the exhaustiveness of side chain sampling made the most significant contribution to the accuracy of pK_a calculations. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

A shift in protein S‐palmitoylation,with persistence of growth‐associated substrates,marks a critical period for synaptic plasticity in developing brain

In the mammalian cortex, the initial formation of synaptic connections is followed by a prolonged period during which synaptic circuits are functional, but retain an elevated capacity for activity‐dependent remodeling and functional plasticity. During this period, synaptic terminals appear fully mature, morphologically and physiologically. We show here, however, that synaptic terminals during this period are distinguished by their simultaneous accumulation of multiple growth‐associated proteins at levels characteristic of axonal growth cones, and proteins involved in synaptic transmitter release at levels characteristic of adult synapses. We show further that newly formed synapses undergo a switch in the dynamic S‐palmitoylation of proteins early in the critical period, which includes a large and specific decrease in the palmitoylation of GAP‐43 and other major substrates characteristic of growth cones. Previous studies have shown that a similar reduction in ongoing palmitoylation of growth cone proteins is sufficient to stop advancing axons in vitro, suggesting that a developmental switch in protein S‐palmitoylation serves to disengage the molecular machinery for axon extension in the absence of local triggers for remodeling during the critical period. Only much later does a decline in the availability of major growth cone components mark the molecular maturation of cortical synapses at the close of the critical period. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 423–437, 1999     

Combination of CD34‐positive cell subsets with infarcted myocardium‐like matrix stiffness: a potential solution to cell‐based cardiac repair

Detection of the optimal cell transplantation strategy for myocardial infarction (MI) has attracted a great deal of attention. Commitment of engrafted cells to angiogenesis within damaged myocardium is regarded as one of the major targets in cell‐based cardiac repair. Bone marrow–derived CD34‐positive cells, a well‐characterized population of stem cells, might represent highly functional endothelial progenitor cells and result in the formation of new blood vessels. Recently, physical microenvironment (extracellular matrix stiffness) around the engrafted cells was found to exert an essential impact on their fate. Stem cells are able to feel and respond to the tissue‐like matrix stiffness to commit to a relevant lineage. Notably, the infarct area after MI experiences a time‐dependent stiffness change from flexible to rigid. Our previous observations demonstrated myocardial stiffness‐dependent differentiation of the unselected bone marrow–derived mononuclear cells (BMMNCs) along endothelial lineage cells. Myocardial stiffness (~42 kPa) within the optimal time domain of cell engraftment (at week 1 to 2) after MI provided a more favourable physical microenvironment for cell specification and cell‐based cardiac repair. However, the difference in tissue stiffness‐dependent cell differentiation between the specific cell subsets expressing and no expressing CD34 phenotype remains uncertain. We presumed that CD34‐positive cell subsets facilitated angiogenesis and subsequently resulted in cardiac repair under induction of infarcted myocardium‐like matrix stiffness compared with CD34‐negative cells. If the hypothesis were true, it would contribute greatly to detect the optimal cell subsets for cell therapy and to establish an optimized therapy strategy for cell‐based cardiac repair.     

O‐GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease

Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)‐CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O‐GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O‐linked N‐acetylglucosamine (O‐GlcNAc). O‐GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O‐GlcNAcylation in metabolically active rat brain slices by O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidenamino) N‐phenylcarbamate (PUGNAc), an inhibitor of N‐acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non‐PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O‐GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O‐GlcNAcylation through intracerebroventricular injection of 6‐diazo‐5‐oxo‐l ‐norleucine (DON), the inhibitor of glutamine fructose‐6‐phosphate amidotransferase, suppressed PKA‐CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O‐GlcNAcylation is a novel mechanism that regulates PKA‐CREB signaling. Downregulation of O‐GlcNAcylation suppresses PKA‐CREB signaling and consequently causes learning and memory deficits in AD.     

A thermodynamic approach to the conformational preferences of the 180–195 segment derived from the human prion protein α2‐helix

On consideration that intrinsic structural weakness could affect the segment spanning the α2‐helical residues 173–195 of the PrP, we have investigated the conformational stabilities of some synthetic Ala‐scanned analogs of the peptide derived from the 180–195 C‐terminal sequence, using a novel approach whose theoretical basis originates from protein thermodynamics. Even though a quantitative comparison among peptides could not be assessed to rank them according to the effect caused by single amino acid substitution, as a general trend, all peptides invariably showed an appreciable preference for an α‐type organization, consistently with the fact that the wild‐type sequence is organized as an α‐helix in the native protein. Moreover, the substitution of whatever single amino acid in the wild‐type sequence reduced the gap between the α‐ and the β‐propensity, invariably enhancing the latter, but in any case this gap was larger than that evaluated for the full‐length α2‐helix‐derived peptide. It appears that the low β‐conformation propensity of the 180–195 region depends on the simultaneous presence of all of the Ala‐scanned residues, indirectly confirming that the N‐terminal 173–179 segment could play a major role in determining the chameleon conformational behavior of the entire 173–195 region in the PrP. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Miklós Bodanszky Award Lecture: Advances in the selective targeting of protein phosphatase‐1 and phosphatase‐2A with peptides

Protein phosphatase‐1 and phosphatase‐2A are two ubiquitously expressed enzymes known to catalyze the majority of dephosphorylation reactions on serine and threonine inside cells. They play roles in most cellular processes and are tightly regulated by regulatory subunits in holoenzymes. Their misregulation and malfunction contribute to disease development and progression, such as in cancer, diabetes, viral infections, and neurological as well as heart diseases. Therefore, targeting these phosphatases for therapeutic use would be highly desirable; however, their complex regulation and high conservation of the active site have been major hurdles for selectively targeting them in the past. In the last decade, new approaches have been developed to overcome these hurdles and have strongly revived the field. I will focus here on peptide‐based approaches, which contributed to showing that these phosphatases can be targeted selectively and aided in rethinking the design of selective phosphatase modulators. Finally, I will give a perspective on www.depod.org , the human dephosphorylation database, and how it can aid phosphatase modulator design. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.