Polyphosphate, a linear polymer of inorganic phosphate, is secreted by activated platelets and accumulates in many infectious microorganisms. We recently showed that polyphosphate modulates the blood coagulation cascade at 3 steps: it triggers the contact pathway, it accelerates factor V activation, and it enhances fibrin polymerization. We now report that polyphosphate exerts differential effects on blood clotting, depending on polymer length. Very long polymers (> 500mers, such as those present in microorganisms) were required for optimal activation of the contact pathway, while shorter polymers (ϳ 100mers, similar to the polymer lengths released by platelets) were sufficient to accelerate factor V activation and abrogate the anticoagulant function of the tissue factor pathway inhibitor. Optimal enhancement of fibrin clot turbidity by polyphosphate required > 250mers. Pyrophosphate, which is also secreted by activated platelets, potently blocked polyphosphate-mediated enhancement of fibrin clot structure, suggesting that pyrophosphate is a novel regulator of fibrin function. In conclusion, polyphosphate of the size secreted by platelets is very efficient at accelerating blood clotting reactions but is less efficient at initiating them or at modulating clot structure. Microbial polyphosphate, which is highly procoagulant, may function in host responses to pathogens. IntroductionPolyphosphate (polyP)-a linear polymer of inorganic phosphateaccumulates in a variety of microorganisms 1 and is secreted by activated human platelets. 2,3 We recently showed that polyP is a potent modulator of the human blood-clotting system. [3][4][5][6] The polymer lengths of polyP are known to vary substantially among different organisms and cell types, with relatively short polymers being secreted by human platelets (ϳ 60-100 phosphate units long) 2,3 and very long polymers accumulating in microorganisms (many hundreds to more than 1000 phosphate units long). 1 In this study, we demonstrate that shorter versus longer polymers of polyP have differential effects on the blood clotting system, with important physiologic/pathophysiologic implications. PolyP has been widely described in unicellular organisms such as bacteria, fungi, algae, and protozoa, where it plays diverse physiologic roles, including regulating growth, stress responses, and virulence. 1,7 Comparatively less is known about the metabolism or physiologic roles of polyP in mammalian cells, 8 although polyP is reported to induce apoptosis in plasma cells, 9 promote calcification in osteoblasts, 10 block metastasis of melanoma cells in a mouse model, 11 and possibly serve as a regulatory factor in proliferative signaling pathways. 12 PolyP is present at high concentrations in dense granules of human platelets and is secreted upon platelet activation. 2,3 PolyP has a half-life in plasma of approximately 90 minutes, because of degradation by phosphatases. 4,13 We recently showed that polyP is a potent hemostatic regulator, acting at 3 points in the blood clotting cascade: it...

Sublancin is shown to be an S-linked glycopeptide containing a glucose attached to a Cys residue, establishing a new post-translational modification. The activity of the S-glycosyl transferase was reconstituted in vitro and the enzyme is shown to have relaxed substrate specificity allowing the preparation of analogs of sublancin. Glycosylation is essential for its antimicrobial activity.

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca 2+ ions. While Ca 2+ is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca 2+ on the structure and dynamics of lipid bilayers. Ca 2+ -induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane-mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing 13 C, 15 Nlabeled PS demonstrate that Ca 2+ ions promote two major PS headgroup conformations, which are well resolved in two-dimensional 13 C-13 C, 15 N-13 C and 31 P-13 C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca 2+ provide an atomic view of the conformational effects underlying the observed spectra.In healthy cells, phosphatidylserine (PS) resides on the inner leaflet of the plasma membrane (1) and represents 10-20% of all plasma membrane lipids (2,3). PS both imparts a negative surface potential for nonspecific binding of cationic proteins (4,5) and recruits several proteins through specific interactions, frequently involving Ca 2+ (6). Externalization of PS in activated platelets and apoptotic cells constitutes a signal eliciting coagulation and † This work was supported by the National Institute of General Medical Sciences, NIH (R01-GM075937 and R01-GM079530 to C.M.R., and R01-GM086749 and R01-GM067887 to E.T.), the National Center for Research Resources, NIH (P41-RR05969 to E.T.), the National Heart Lung and Blood Institute, NIH (R01 HL47014 to J.H.M. and R01 HL103999 to J.H.M. and C.M.R.), and by the American Heart Association (0920045G to R.D.H.). * To whom correspondence should be addressed: Chad Rienstra, Dept. of Chemistry, University of Illinois at Urbana-Champaign, 600 S Mathews Ave, Box 50-6, Urbana, IL 61801, Phone: 217-244-4655. Fax: 217-244-3186. rienstra@scs.uiuc.edu. # These two authors contributed equally to this work. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2012 March 29. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript phagocytosis, respectively (7,8). It is well documented that relatively high concentrations of Ca 2+ can exert dramatic effects on membranes contain...

The study of micro-or nanocrystalline proteins by magic-angle spinning (MAS) solid-state NMR (SSNMR) gives atomic-resolution insight into structure in cases when single crystals cannot be obtained for diffraction studies. Subtle differences in the local chemical environment around the protein, including the characteristics of the co-solvent and the buffer, determine whether a protein will form single crystals. The impact of these small changes in formulation is also evident in the SSNMR spectra, but leads only to correspondingly subtle changes in the spectra. Here we demonstrate that several formulations of GB1 microcrystals yield very high-quality SSNMR spectra, although only a subset of conditions enable growth of single crystals. We have characterized these polymorphs by X-ray powder diffraction and assigned the SSNMR spectra. Assignments of the 13 C and 15 N SSNMR chemical shifts confirm that the backbone structure is conserved, indicative of a common protein fold, but sidechain chemical shifts are changed on the surface of the protein, in a manner dependent upon crystal packing and electrostatic interactions with salt in the mother liquor. Our results demonstrate the ability of SSNMR to reveal minor structural differences among crystal polymorphs. This ability has potential practical utility for studying formulation chemistry of industrial and therapeutic proteins, as well as for deriving fundamental insights into the phenomenon of single crystal growth.

Even as available magnetic fields for NMR continue to increase, resolution remains one of the most critical limitations in assigning and solving structures of larger biomolecules. Here we present a novel constant-time through-bond correlation spectroscopy for solids that offers superior resolution for 13C chemical shift assignments in proteins. In this experiment, the indirect evolution and transfer periods are combined into a single constant time interval, offering increased resolution while not sacrificing sensitivity. In GB1, this allows us to resolve peaks that are otherwise unresolved and to make assignments in the absence of multibond transfers.

␣-Synuclein (AS) is an intrinsically unstructured protein in aqueous solution but is capable of forming ␤-sheet-rich fibrils that accumulate as intracytoplasmic inclusions in Parkinson disease and certain other neurological disorders. However, AS binding to phospholipid membranes leads to a distinct change in protein conformation, stabilizing an extended amphipathic ␣-helical domain reminiscent of the exchangeable apolipoproteins. To better understand the significance of this conformational change, we devised a novel bacteriophage display screen to identify protein binding partners of helical AS and have identified 20 proteins with roles in diverse cellular processes related to membrane trafficking, ion channel modulation, redox metabolism, and gene regulation. To verify that the screen identifies proteins with specificity for helical AS, we further characterized one of these candidates, endosulfine ␣ (ENSA), a small cAMP-regulated phosphoprotein implicated in the regulation of insulin secretion but also expressed abundantly in the brain. We used solution NMR to probe the interaction between ENSA and AS on the surface of SDS micelles. Chemical shift perturbation mapping experiments indicate that ENSA interacts specifically with residues in the N-terminal helical domain of AS in the presence of SDS but not in aqueous buffer lacking SDS. The ENSA-related protein ARPP-19 (cAMP-regulated phosphoprotein 19) also displays specific interactions with helical AS. These results confirm that the helical N terminus of AS can mediate specific interactions with other proteins and suggest that membrane binding may regulate the physiological activity of AS in vivo.

Most steps of the blood clotting cascade require the assembly of a serine protease with its specific regulatory protein on a suitable phospholipid bilayer. Unfortunately, the molecular details of how blood clotting proteins bind to membrane surfaces remain poorly understood, owing to a dearth of techniques for studying protein-membrane interactions at high resolution. Our laboratories are tackling this question using a combination of approaches, including nanoscale membrane bilayers, solid-state NMR, and large-scale molecular dynamics simulations. These studies are now providing structural insights at atomic resolution into clotting protein-membrane interactions.

Endosulfine-alpha (ENSA) is a 121-residue cAMP-regulated phosphoprotein, originally identified as an endogenous regulator of ATP-sensitive potassium channels. ENSA has been implicated in the regulation of insulin secretion, and expression of ENSA is decreased in brains of both Alzheimer's disease (AD) and Down's syndrome patients. We recently described membrane-dependent interactions between ENSA and the Parkinson's disease associated protein alpha-synuclein. Here we characterize the conformational change in ENSA that occurs upon binding to membranes. Secondary chemical shift analysis demonstrates formation of four helices in the lipid-bound state that are not present in the absence of lipid. The helical structure is maintained in several different lipid mimetics (sodium dodecyl sulfate, dodecyl phosphocholine, lyso 1-palmitoyl phosphatidylglycerol, and phospholipid vesicles). Introduction of a mutation (S109E) to mimic PKA phosphorylation of ENSA leads to a perturbation of the fourth helix and disrupts the interaction with alpha-synuclein. These data establish ENSA as an intrinsically unstructured protein that adopts a stable structure upon membrane binding, properties it shares with its binding partner alpha-synuclein.