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The blood protein plasminogen is proteolytically cleaved to produce angiostatin and kringle 5 (K5), both of which are known angiogenesis inhibitors. A common structural element between K5, angiostatin and other endogenous angiogenesis inhibitors is the presence of the kringle protein-interacting domain. Another kringle domain-containing protein, hepatocyte growth factor (HGF), promotes angiogenesis by binding to and stimulating the tyrosine kinase receptor Met. HGF binding to Met is dependent on the kringle domains of HGF. Because both K5 and HGF contain kringle motifs and because these proteins have opposite effects on angiogenesis, we hypothesised that K5 can antagonise HGF-mediated signalling in a Met-dependent manner. We determined that K5 binding to H1299 cells is competed by HGF suggesting that these two proteins bind to the same protein. Purified K5 immunoprecipitates with Met and this interaction is abolished by increasing doses of HGF. Using proliferation, phosphorylation of Met and Akt as markers of HGF activity, we determined that K5 inhibits HGF-mediated signalling. Taken together, these data support a model by which K5 binds to Met and functions as a competitive antagonist of HGF signalling and presents a novel mechanism of action of K5. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies comprise a major class of biologic therapeutics and are also extensively studied in immunology. Given the importance of glycans on antibodies, fluorescent labeling of enzymatically released glycans and their LC/MS analysis is routinely used for in-depth characterization of antibody glycosylation. In this technical note, we propose a method for facile characterization of glycans in the variable region of antibodies using sequential enzymatic digests with Endoglycosidase-S2 and RapidTM Peptide-N-Glycosidase-F followed by labeling with a fluorescent dye carrying an NHS-carbamate moiety. The results and proposed mechanism also suggest that the choice of glycosidases along with the labeling chemistry is critical for accurate glycan analysis for a desired application., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To investigate the role played by the unique pre-DFG residue Val 195 of Cdc7 kinase on the potency of azaindole-chloropyridines (1), a series of novel analogues with various chloro replacements were synthesized and evaluated for their inhibitory activity against Cdc7. X-ray cocrystallization using a surrogate protein, GSK3β, and modeling studies confirmed the azaindole motif as the hinge binder. Weaker hydrophobic interactions with Met 134 and Val 195 by certain chloro replacements (e.g., H, methyl) led to reduced Cdc7 inhibition. Meanwhile, data from other replacements (e.g., F, O) indicated that loss of such hydrophobic interaction could be compensated by enhanced hydrogen bonding to Lys 90. Our findings not only provide an in-depth understanding of the pre-DFG residue as another viable position impacting kinase inhibition, they also expand the existing knowledge of ligand-Cdc7 binding.

Successfully forming ligand-protein complexes with specific compounds can be a significant challenge in supporting structure-based drug design for a given protein target. In this respect, an on-column ligand- and detergent-exchange method was developed to obtain ligand-protein complexes of an adamantane series of compounds with 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) after a variety of other complexation methods had failed. This report describes the on-column exchange method and an unexpected byproduct of the method in which artificial trimers were observed in the structures.

Purpose: PARP inhibitors are being developed as therapeutic agents for cancer. More than six compounds have entered clinical trials. The majority of these compounds are β-nicotinamide adenine dinucleotide (NAD(+))-competitive inhibitors. One exception is iniparib, which has been proposed to be a noncompetitive PARP inhibitor. In this study, we compare the biologic activities of two different structural classes of NAD(+)-competitive compounds with iniparib and its C-nitroso metabolite., Experimental Design: Two chemical series of NAD(+)-competitive PARP inhibitors, iniparib and its C-nitroso metabolite, were analyzed in enzymatic and cellular assays. Viability assays were carried out in MDA-MB-436 (BRCA1-deficient) and DLD1(-/-) (BRCA2-deficient) cells together with BRCA-proficient MDA-MB-231 and DLD1(+/+) cells. Capan-1 and B16F10 xenograft models were used to compare iniparib and veliparib in vivo. Mass spectrometry and the (3)H-labeling method were used to monitor the covalent modification of proteins., Results: All NAD(+)-competitive inhibitors show robust activity in a PARP cellular assay, strongly potentiate the activity of temozolomide, and elicit robust cell killing in BRCA-deficient tumor cells in vitro and in vivo. Cell killing was associated with an induction of DNA damage. In contrast, neither iniparib nor its C-nitroso metabolite inhibited PARP enzymatic or cellular activity, potentiated temozolomide, or showed activity in a BRCA-deficient setting. We find that the nitroso metabolite of iniparib forms adducts with many cysteine-containing proteins. Furthermore, both iniparib and its nitroso metabolite form protein adducts nonspecifically in tumor cells., Conclusions: Iniparib nonselectively modifies cysteine-containing proteins in tumor cells, and the primary mechanism of action for iniparib is likely not via inhibition of PARP activity., (©2011 AACR.)

The DVD-Ig (TM) protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.

The HIV protease inhibitor ritonavir (RTV) is also a potent inhibitor of the metabolizing enzyme cytochrome P450 3A (CYP3A) and is clinically useful in HIV therapy in its ability to enhance human plasma levels of other HIV protease inhibitors (PIs). A novel series of CYP3A inhibitors was designed around the structural elements of RTV believed to be important to CYP3A inhibition, with general design features being the attachment of groups that mimic the P2-P3 segment of RTV to a soluble core. Several analogs were found to strongly enhance plasma levels of lopinavir (LPV), including 8, which compares favorably with RTV in the same model. Interestingly, an inverse correlation between in vitro inhibition of CYP3A and elevation of LPV was observed. The compounds described in this study may be useful for enhancing the pharmacokinetics of drugs that are metabolized by CYP3A.

Alzheimer's disease (AD) is a neurodegenerative disorder that is linked to the presence of amyloid beta-peptides that can form insoluble fibrils or soluble oligomeric assemblies. Soluble forms are present in the brains and tissues of Alzheimer's patients, and their presence correlates with disease progression. Long-lived soluble forms can be generated in vitro by using small amounts of aliphatic hydrocarbon chains of detergents or fatty acids in preparations of amyloid beta-peptides. Using NMR, we have characterized soluble oligomers of Abeta preglobulomer and globulomer that are stable and alter synaptic activity. The NMR data indicate that these soluble forms have a mixed parallel and antiparallel beta-sheet structure that is different from fibrils which contain only parallel beta-sheets. Using the structural data, we engineered a disulfide bond into the soluble Abeta globulomer to give a "new" soluble antigen that is stable, homogeneous, and binds with the same affinity to selective antibodies as the parent wt globulomer.

Recombinant human erythropoietin (rHu-EPO) is used to treat anemia by activating the erythropoietin receptor (EPOR) in erythroid progenitor cells, leading to proliferation and differentiation into mature red blood cells. To allow less frequent dosing, a hyperglycosylated version of EPO has been developed with a longer half-life. In principle, an agonistic antibody targeting EPOR would offer an even longer half-life, support robust monthly dosing, and, unlike EPO products, reduce the risk of pure red cell aplasia. The efficiency of signaling and corresponding potency of previously reported antibody mimics are generally suboptimal compared with EPO and not suitable for clinical use. Here we describe a potent, fully human, agonistic antibody (ABT007) targeting EPOR that supports potent, more sustained, and less pulsatile elevation of hematocrit in a human EPOR-expressing transgenic mouse model compared with standard doses of rHu-EPO while requiring less frequent dosing. Resolution of the crystal structure of the EPOR extracellular domain (ECD) complexed to the ABT007 Fab fragment, determined at 0.32 nm, identifies a binding site that is consistent with a novel mechanism of receptor activation based on a unique antibody-imposed conformational change. These results demonstrate that a symmetric molecule can serve as a potent activator of the EPOR.

As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor. Although further studies are needed to conclusively establish the mode of action of the biphenyl series, these compounds represent novel leads that can serve as the basis for the development of novel antibacterial agents that appear to work via an unprecedented mechanism of action. Overall, these results support the genomics-driven hypothesis that targeting bacterial essential gene products that are not present in eukaryotic cells can identify novel antibacterial agents.

Two new proteins of approximately 70 amino acids in length, corresponding to an unnaturally-linked N- and C-helix of the ectodomain of the gp41 protein from the human immunodeficiency virus (HIV) type 1, were designed and characterized. A designed tripeptide links the C-terminus of the C-helix with the N-terminus of the N-helix in a circular permutation so that the C-helix precedes the N-helix in sequence. In addition to the artificial peptide linkage, the C-helix is truncated at its N-terminus to expose a region of the N-helix known as the "Trp-Trp-Ile" binding pocket. Sedimentation, crystallographic, and nuclear magnetic resonance studies confirmed that the protein had the desired trimeric structure with an unoccupied binding site. Spectroscopic and centrifugation studies demonstrated that the engineered protein had ligand binding characteristics similar to previously reported constructs. Unlike previous constructs which expose additional, shallow, non-conserved, and undesired binding pockets, only the single deep and conserved Trp-Trp-Ile pocket is exposed in the proteins of this study. This engineered version of gp41 protein will be potentially useful in research programs aimed at discovery of new drugs for therapy of HIV-infection in humans.

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.

Ras mutation has been detected in approximately 20-30% of all human carcinomas, primarily in pancreatic, colorectal, lung and bladder carcinomas. The indirect inhibition of Ras activity by inhibiting farnesyltransferase (FTase) function is one therapeutic intervention to control tumor growth. Here we report the preclinical anti-tumor activity of our most advanced FTase inhibitor (FTI), ABT-100, and a direct comparison with the current clinical candidates. ABT-100 is a highly selective, potent and orally bioavailable FTI. It broadly inhibits the growth of solid tumors in preclinical animal models. Thus, ABT-100 is an attractive candidate for further clinical evaluation. In addition, our results provide plausible insights to explain the impressive potency and selectivity of ABT-100. Finally, we have demonstrated that ABT-100 significantly suppresses the expression of vascular endothelial growth factor (VEGF) mRNA and secretion of VEGF protein, as well as inhibiting angiogenesis in the animal model.

Potent inhibitors of 7,8-dihydroneopterin aldolase (DHNA; EC 4.1.2.25) have been discovered using CrystaLEAD X-ray crystallographic high-throughput screening followed by structure-directed optimization. Screening of a 10 000 compound random library provided several low affinity leads and their corresponding X-ray crystal structures bound to the enzyme. The presence of a common structural feature in each of the leads suggested a strategy for the construction of a directed library of approximately 1000 compounds that were screened for inhibitory activity in a traditional enzyme assay. Several lead compounds with IC(50) values of about 1 microM against DHNA were identified, and crystal structures of their enzyme-bound complexes were obtained by cocrystallization. Structure-directed optimization of one of the leads thus identified afforded potent inhibitors with submicromolar IC(50) values.

We describe a new approach to affinity selection based on the application of centrifugal force to macromolecules in solution. The method relies on the well known macromolecular hydrodynamic principles of centrifugation. It can be automated and operated in a centralized fashion, or it can be decentralized and used by single researchers or networks of researchers with a minimal additional capital investment. In this method, a centrifugal driving force is used to establish a differential and selective concentration gradient between a therapeutic target and potential ligands in compound libraries. This concentration gradient, in turn, drives the binding of ligands. Once formed, the differential concentration gradient of target macromolecules and ligands is fractionated to capture the self-sorting binding events. Ligand binding is defined by the individual ligand binding constants, so tight binding ligands will essentially distribute identically with the protein target, and weaker binding ligands will not. The level of affinity needed to operationally define tight binding can be adjusted by selecting the initial concentration conditions or centrifugal force. A variety of rapid, commonly available, detection methods can be used to assess binding in the fractionated samples. The method can be broadly applied in drug discovery efforts to examine most types of cell-cell, protein-protein, and protein-small molecule interactions. We describe the application of this method to systems of small molecule interactions with several macromolecules of therapeutic interest.

The Bcl-2 family of proteins play a pivotal role in the regulation of programmed cell death. One of the postulated mechanisms for the function of these proteins involves the formation of ion channels in membranes. As a first step to structurally characterize these proteins in a membrane environment, we investigated the structure of a Bcl-x(L) mutant protein when incorporated into small detergent micelles. This form of Bcl-x(L) lacks the loop (residues 49-88) between helix 1 and helix 2 and the putative C-terminal transmembrane helix (residues 214-237). Below the critical micelle concentration (CMC), Bcl-x(L) binds detergents in the hydrophobic groove that binds to pro-apoptotic proteins. However, above the CMC, Bcl-x(L) undergoes a dramatic conformational change. Using NMR methods, we characterized the secondary structure of Bcl-x(L) in the micelle-bound form. Like Bcl-x(L) in aqueous solution, the structure of the protein when dissolved in dodecylphosphocholine (DPC) micelles consists of several alpha-helices separated by loops. However, the length and position of the individual helices of Bcl-x(L) in micelles differ from those in aqueous solution. The location of Bcl-x(L) within the micelle was examined from the analysis of protein-detergent NOEs and limited proteolysis. In addition, the mobility of the micelle-bound form of Bcl-x(L) was investigated from NMR relaxation measurements. On the basis of these studies, a model is proposed for the structure, dynamics, and location of Bcl-x(L) in micelles. In this model, Bcl-x(L) has a loosely packed, dynamic structure in micelles, with helices 1 and 6 and possibly helix 5 partially buried in the hydrophobic interior of the micelle. Other parts of the protein are located near the surface or on the outside of the micelle.

bcl-x is a member of the bcl-2 family of genes. The major protein product, Bcl-x(L), is a 233-amino-acid protein which has antiapoptotic properties. In contrast, one of the alternatively spliced transcripts of the bcl-x gene codes for the protein Bcl-x(S), which lacks 63 amino acids present in Bcl-x(L) and has proapoptotic activity. Unlike other proapoptotic Bcl-2 family members, such as Bax and Bak, Bcl-x(S) does not seem to induce cell death in the absence of an additional death signal. However, Bcl-x(S) does interfere with the ability of Bcl-x(L) to antagonize Bax-induced death in transiently transfected 293 cells. Mutational analysis of Bcl-x(S) was conducted to identify the domains necessary to mediate its proapoptotic phenotype. Deletion mutants of Bcl-x(S) which still contained an intact BH3 domain retained the ability to inhibit survival through antagonism of Bcl-x(L). Bcl-x(S) was able to form heterodimers with Bcl-x(L) in mammalian cells, and its ability to inhibit survival correlated with the ability to heterodimerize with Bcl-x(L). Deletion mutants of Bax and Bcl-2, which lacked BH1 and BH2 domains but contained a BH3 domain, were able to antagonize the survival effect conferred by Bcl-x(L). The results suggest that BH3 domains from both pro- and antiapoptotic Bcl-2 family members, while lacking an intrinsic ability to promote programmed cell death, can be potent inhibitors of Bcl-x(L) survival function.

Guanine nucleotide exchange factors for the Rho family of GTPases contain a Dbl homology (DH) domain responsible for catalysis and a pleckstrin homology (PH) domain whose function is unknown. Here we describe the solution structure of the N-terminal DH domain of Trio that catalyzes nucleotide exchange for Rac1. The all-alpha-helical protein has a very different structure compared to other exchange factors. Based on site-directed mutagenesis, functionally important residues of the DH domain were identified. They are all highly conserved and reside in close proximity on two a helices. In addition, we have discovered a unique capability of the PH domain to enhance nucleotide exchange in DH domain-containing proteins.

The Bcl-2 related protein Bad is a promoter of apoptosis and has been shown to dimerize with the anti-apoptotic proteins Bcl-2 and Bcl-XL. Overexpression of Bad in murine FL5.12 cells demonstrated that the protein not only could abrogate the protective capacity of coexpressed Bcl-XL but could accelerate the apoptotic response to a death signal when it was expressed in the absence of exogenous Bcl-XL. Using deletion analysis, we have identified the minimal domain in the murine Bad protein that can dimerize with Bcl-xL. A 26-amino-acid peptide within this domain, which showed significant homology to the alpha-helical BH3 domains of related apoptotic proteins like Bak and Bax, was found to be necessary and sufficient to bind Bcl-xL. To determine the role of dimerization in regulating the death-promoting activity of Bad and the death-inhibiting activity of Bcl-xL, mutations within the hydrophobic BH3-binding pocket in Bcl-xL that eliminated the ability of Bcl-xL to form a heterodimer with Bad were tested for the ability to promote cell survival in the presence of Bad. Several of these mutants retained the ability to impart protection against cell death regardless of the level of coexpressed Bad protein. These results suggest that BH3-containing proteins like Bad promote cell death by binding to antiapoptotic members of the Bcl-2 family and thus inhibiting their survival promoting functions.

The adapter protein Shc is a critical component of mitogenic signaling pathways initiated by a number of receptors. Shc can directly bind to several tyrosine-phosphorylated receptors through its phosphotyrosine-binding (PTB) domain, and a role for the PTB domain in phosphotyrosine-mediated signaling has been well documented. The structure of the Shc PTB domain demonstrated a striking homology to the structures of pleckstrin homology domains, which suggested acidic phospholipids as a second ligand for the Shc PTB domain. Here we demonstrate that Shc binding via its PTB domain to acidic phospholipids is as critical as binding to phosphotyrosine for leading to Shc phosphorylation. Through structure-based, targeted mutagenesis of the Shc PTB domain, we first identified the residues within the PTB domain critical for phospholipid binding in vitro. In vivo, the PTB domain was essential for localization of Shc to the membrane, as mutant Shc proteins that failed to interact with phospholipids in vitro also failed to localize to the membrane. We also observed that PTB domain-dependent targeting to the membrane preceded the PTB domain's interaction with the tyrosine-phosphorylated receptor and that both events were essential for tyrosine phosphorylation of Shc following receptor activation. Thus, Shc, through its interaction with two different ligands, is able to accomplish both membrane localization and binding to the activated receptor via a single PTB domain.

Heterodimerization between members of the Bcl-2 family of proteins is a key event in the regulation of programmed cell death. The molecular basis for heterodimer formation was investigated by determination of the solution structure of a complex between the survival protein Bcl-xL and the death-promoting region of the Bcl-2-related protein Bak. The structure and binding affinities of mutant Bak peptides indicate that the Bak peptide adopts an amphipathic alpha helix that interacts with Bcl-xL through hydrophobic and electrostatic interactions. Mutations in full-length Bak that disrupt either type of interaction inhibit the ability of Bak to heterodimerize with Bcl-xL.

THE Bcl-2 family of proteins regulate programmed cell death by an unknown mechanism. Here we describe the crystal and solution structures of a Bcl-2 family member, Bcl-xL (ref. 2). The structures consist of two central, primarily hydrophobic alpha-helices, which are surrounded by amphipathic helices. A 60-residue loop connecting helices alpha1 and alpha2 was found to be flexible and non-essential for anti-apoptotic activity. The three functionally important Bcl-2 homology regions (BH1, BH2 and BH3) are in close spatial proximity and form an elongated hydrophobic cleft that may represent the binding site for other Bcl-2 family members. The arrangement of the alpha-helices in Bcl-xL is reminiscent of the membrane translocation domain of bacterial toxins, in particular diphtheria toxin and the colicins. The structural similarity may provide a clue to the mechanism of action of the Bcl-2 family of proteins.

The adaptor protein Shc has been implicated in Ras signaling via association with many tyrosine-phosphorylated receptors, including growth factor receptors, antigen receptors on T and B cells, and cytokine receptors. Shc could interact with the activated receptors through the carboxyl-terminal Src homology 2 (SH2) domain or the structurally unrelated amino-terminal phosphotyrosine binding (PTB) domain. Using NMR and surface plasmon resonance techniques, we have measured the binding affinities of the SH2 and the PTB domains of Shc to a series of phosphotyrosine-containing peptides derived from known Shc binding sites. Tyrosine-phosphorylated peptides derived from Trk (pY490), polyoma virus middle T-antigen (pY250), ErbB3 (pY1309), and epidermal growth factor receptor (pY1086, pY1148, and pY1114) that contain NPXpY sequences bind preferentially to the PTB domain of Shc with Kd values of 0.02-5.3 microM. The binding affinities of these peptides to the Shc SH2 domain were in the range of 220-1290 microM. In contrast, tyrosine-phosphorylated peptides from epidermal growth factor receptor (pY992, pY1173) and the zeta chain of the T-cell receptor bind preferentially to the SH2 domain (Kd = 50-130 microM) versus the PTB domain (Kd > 680 microM). From these studies, the relative contribution of the individual domains of Shc for binding to the phosphotyrosine-containing portions of these proteins was determined. In addition, our data indicate that the high affinity binding of the PTB domain to the NPXpY-containing peptides results from a very high association rate and a rapid dissociation rate, which is similar to previous results observed for the SH2-phosphopeptide complexes.

The nuclear magnetic resonance structure of the phosphotyrosine binding (PTB) domain of Shc complexed to a phosphopeptide reveals an alternative means of recognizing tyrosine-phosphorylated proteins. Unlike in SH2 domains, the phosphopeptide forms an antiparallel beta-strand with a beta-sheet of the protein, interacts with a hydrophobic pocket through the (pY-5) residue, and adopts a beta-turn. The PTB domain is structurally similar to pleckstrin homology domains (a beta-sandwich capped by an alpha-helix) and binds to acidic phospholipids, suggesting a possible role in membrane localization.

The pleckstrin homology (PH) domain is a protein module of approximately 100 amino acids that is found in several proteins involved in signal transduction [for a recent review, see Gibson et al. (1994) Trends Biochem. Sci. 19, 349-353]. Although the specific function of the PH domain has not yet been elucidated, many of the proteins which contain this domain associate with phospholipid membranes, and PH domains have been shown to bind to phosphatidylinositol 4,5-bisphosphate (PIP2) [Harlan et al. (1994) Nature 371, 168-170] and the beta gamma subunits of G-proteins [Touhara et al. (1994) J. Biol. Chem. 269, 10217-10220]. We have postulated that pleckstrin homology domains may be important for the translocation of proteins to the membrane by an interaction with the negatively charged head group of phospholipids. Here we show the importance of three conserved lysine residues for binding to PIP2 by site-directed mutagenesis. These results should aid future site-directed mutagenesis studies in probing the function of PIP2-PH domain interactions in the various proteins containing this module. In addition, we examine the specificity of this binding and illustrate the importance of charge--charge interactions in PIP2-PH domain complex formation from binding experiments involving PIP2 analogs.

A method for the calibration of size-exclusion chromatographic columns is proposed that takes into account the nonlinear dependence of the Stokes radius Rs upon the partition coefficient KD. The method is based on the assumption that the pore size distribution of a molecular sieve column can be described by the sum of two Gaussian distributions and has been successfully tested with low-pressure chromatography gels (Sephacryl and Superose) and HPLC gels (TSK SW) over a wide range of protein sizes. An application of this method is described, in which aggregation states of the membrane protein prostaglandin H2 synthase solubilized in nonionic detergents are estimated.

The pleckstrin homology (PH) domain is a new protein module of around 100 amino acids found in several proteins involved in signal transduction. Although its specific function has yet to be elucidated, the carboxy-terminal regions of many PH domains bind to the beta gamma subunits of G proteins. On the basis of structural similarities between PH domains and lipid-binding proteins, we have proposed that PH domains may be binding to lipophilic molecules. Indeed, many of the proteins that contain this domain associate with phospholipid membranes, and disruption of this domain can interfere with membrane association. Here we report that PH domains bind to phosphatidylinositol-4,5-bisphosphate and show that the lipid-binding site is located at the lip of the beta-barrel. This suggests that PH domains may be important for membrane localization of proteins through interactions with phosphatidylinositol-4,5-bisphosphate.

Rubella hemagglutination inhibition antibody titer was determined for 702 sixth grade students from nine randomly chosen schools in a community served well medically. Rubella immunization had been given by private physicians, the health department, hospital clinics, and through a mass immunization effort in 1970. The overall susceptibility rate, as defined by a rubella hemagglutination inhibition titer of less than 1:8, was 15%. Susceptibility did not differ significantly in regard to sex, race, or source of vaccine. Of 469 children with a documented rubella immunization, 13.2% were susceptible or vaccine failures. Menarche was reported by 30% of the girls. To increase the level of protection against rubella during the childbearing years, continued emphasis on early childhood immunization combined with consideration of a booster rubella immunization for preadolescents is recommended.

Serological tests for Rocky Mountain spotted fever were performed on single serum specimens from sixth graders from Forsyth County, North Carolina, an area highly endemic for this disease. Five of 508 *.098%) sera were positive (titer greater than or equal to 1:64) using the indirect fluorescent antibody method. Elevated Proteus agglutination antibody titers (greater than 1:160) to OX-19, but not ot OX-2, were common (19.6%) in these children. No correlation of those with positive OX-19 titers could be made with the cases demonstrating elevated indirect fluorescent antibody titers or with a history of urinary tract infection. These data suggest that the increased prevalence of elevated Proteus OX-19 antibody levels makes a single high titer unreliable in the diagnosis of Rocky Mountain spotted fever and that naturally acquired mild or asymptomatic subclinical cases of Rickettsia rickttsii infection occur.