Abbreviations used in this paper: ADF-H, actin-depolymerizing factor homology; NBD, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole; WH2, WASP homology 2.The online version of this paper contains supplemental material. IntroductionPolymerization of actin fi laments against membranes produces pushing forces that are required for various cellular processes such as motility, morphogenesis, and endocytosis ( Pollard and Borisy, 2003 ;Kaksonen et al., 2006 ). Despite the large number of proteins regulating actin dynamics, many of them interact with actin through a relatively small number of protein domains. Among the central actin-binding domains is the actin-depolymerizing factor homology (ADF-H) domain, which occurs in five functionally distinct classes of proteins: ADF/cofilin, twinfi lin, Abp1/drebrin, coactosin, and glia maturation factor ( Paavilainen et al., 2007 ).The founding member of this family, ADF/cofi lin, binds both monomeric and fi lamentous actin, preferably in the ADPbound form, and induces a structural rearrangement in the actin fi lament that leads to its disassembly. When bound to an actin monomer, ADF/cofi lin inhibits spontaneous nucleotide exchange ( Carlier et al., 1997 ;Bamburg, 1999 ;Andrianantoandro and Pollard, 2006 ). In cells, ADF/cofi lin plays an essential role in various processes by promoting disassembly of aged actin fi laments ( Okreglak and Drubin, 2007 ). In contrast to ADF/cofi lin, which consists of a single ADF-H domain, twinfi lin is composed of two ADF-H domains separated by a short linker region . Twinfi lin binds ADP-actin monomers and fi lament barbed ends with high affi nity, and prevents monomer assembly into fi lament ends Helfer et al., 2006 ). In addition, yeast twinfi lin induces fi lament severing at a low pH ( Moseley et al., 2006 ). Biochemical studies suggested that during barbedend capping, twinfi lin ' s N-terminal ADF-H domain interacts with the terminal actin subunit, whereas the C-terminal ADF-H domain binds to the side of an actin fi lament through a similar mechanism to that of ADF/cofi lin ( Paavilainen et al., 2007 ). The exact functions of the Abp1/drebrin, coactosin, and glia maturation factor are less well understood, although also these proteins are linked to regulation of actin dynamics ( de Hostos et al., 1993 ;Quintero-Monzon et al., 2005 ;Ikeda et al., 2006 ).Although the biochemical activities and cellular functions of ADF-H domain proteins are rapidly being uncovered, the structure of an ADF-H domain in complex with actin has not been reported. Indirect structural methods have provided controversial results, and even the binding site of this domain on actin is not known ( Wriggers et al., 1998 ;Kamal et al., 2007 ). Consequently, the structural mechanisms by which twinfi lin and ADF/cofi lin inhibit nucleotide exchange on actin monomers and how ADF/cofi lin induces fi lament depolymerization/ severing are unknown.A ctin dynamics provide the driving force for many cellular processes including motility and endocytosis. Among the central cytoskeletal re...

Understanding the driving forces underlying molecular recognition is of fundamental importance in chemistry and biology. The challenge is to unravel the binding thermodynamics into separate contributions and to interpret these in molecular terms. Entropic contributions to the free energy of binding are particularly difficult to assess in this regard. Here we pinpoint the molecular determinants underlying differences in ligand affinity to the carbohydrate recognition domain of galectin-3, using a combination of isothermal titration calorimetry, X-ray crystallography, NMR relaxation, and molecular dynamics simulations followed by conformational entropy and grid inhomogeneous solvation theory (GIST) analyses. Using a pair of diastereomeric ligands that have essentially identical chemical potential in the unbound state, we reduced the problem of dissecting the thermodynamics to a comparison of the two protein–ligand complexes. While the free energies of binding are nearly equal for the R and S diastereomers, greater differences are observed for the enthalpy and entropy, which consequently exhibit compensatory behavior, ΔΔ H°(R – S) = −5 ± 1 kJ/mol and −TΔΔ S°(R – S) = 3 ± 1 kJ/mol. NMR relaxation experiments and molecular dynamics simulations indicate that the protein in complex with the S-stereoisomer has greater conformational entropy than in the R-complex. GIST calculations reveal additional, but smaller, contributions from solvation entropy, again in favor of the S-complex. Thus, conformational entropy apparently dominates over solvation entropy in dictating the difference in the overall entropy of binding. This case highlights an interplay between conformational entropy and solvation entropy, pointing to both opportunities and challenges in drug design.

We have solved the structures of mammalian mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved dopamine neurotrophic factor (CDNF). CDNF protects and repairs midbrain dopaminergic neurons in vivo; MANF supports their survival in culture and is also cytoprotective against endoplasmic reticulum (ER) stress. Neither protein structure resembles any known growth factor but the N-terminal domain is a saposin-like lipid-binding domain. MANF and CDNF may thus bind lipids or membranes. Consistent with this, there are two patches of conserved lysines and arginines. The natively unfolded MANF C-terminus contains a CKGC disulphide bridge, such as reductases and disulphide isomerases, consistent with a role in ER stress response. The structure thus explains why MANF and CDNF are bifunctional; neurotrophic activity may reside in the N-terminal domain and ER stress response in the C-terminal domain. Finally, we identified three changes, (MANF)I10-->K(CDNF), (MANF)E79-->M(CDNF) and (MANF)K88-->L(CDNF), that may account for the biological differences between the proteins.

We report the first crystal structures of a family II pyrophosphatase complexed with a substrate analogue, imidodiphosphate (PNP). These provide new insights into the catalytic reaction mechanism of this enzyme family. We were able to capture the substrate complex both by fluoride inhibition and by site-directed mutagenesis providing complementary snapshots of the Michaelis complex. The universally present inorganic pyrophosphatase (PPase, EC 3.6.1.1) 4 is a central enzyme of phosphorus metabolism.PPases are essential enzymes, because they hydrolyze the inorganic pyrophosphate (PP i ) generated during a number of ATPdependent cellular processes and thus provide the necessary thermodynamic pull for them (1). PPases require divalent metal cations for catalysis. Soluble PPases comprise two families, which differ completely in both sequence (2, 3) and structure (4, 5). Family I PPases (reviewed in Ref. 6) occur in all types of cells from bacteria to man, whereas family II PPases occur almost exclusively in bacteria. Of the 57 known family II PPases, 53 occur in eubacteria, 3 in archaebacteria, and 1 in a unicellular eukaryote (Giardia lamblia). Three Vibrio species, including Vibrio cholerae, have genes for both family I and family II PPases. The frequent occurrence of family II PPase in human pathogens (e.g. Streptococcus agalactiae causes neonatal pneumonia, sepsis, and meningitis; Streptococcus mutans, dental caries; and V. cholerae, cholera) makes studies of this enzyme medically important. In addition, family II PPases belong to the "DHH" family of phosphoesterases, named after the characteristic DHH amino acid signature (7). All of these enzymes have similar structures (5, 8) and presumably related catalytic mechanisms.In contrast to family I PPases, which have a simple cup-like single-domain structure, family II PPases have two domains, with the active site at the domain interface (4, 5) (Fig. 1A). The C-terminal domain of family II PPase contains the high affinity substrate-binding site, whereas the catalytic site that binds the nucleophile-coordinating metal cations is located in the N-terminal domain. Closure of the C-terminal domain onto the N-terminal one creates the catalytically competent conformation by bringing the electrophilic phosphate of substrate into the catalytic site (9). The trigger for domain closure is substrate binding to the C-terminal domain in the open conformation (9).The two PPase families catalyze the hydrolysis of PP i in what initially appeared to be similar active sites (4, 5) (Fig. 1B), but their functional properties are significantly different. The natural metal cofactor of family I PPases is Mg 2ϩ , binding to the enzyme with micromolar affinity, whereas family II enzymes are best activated by Mn 2ϩ or Co 2ϩ , which bind with nanomolar affinity. With these metal ions, family II PPases are ϳ10-fold more active than family I PPases (k cat of 1700 -3300 s Ϫ1 versus 110 -330 s Ϫ1 ) (10 -12). Interestingly, Mg 2ϩ confers lower activity but greater substrate-binding affinity on fam...