Structural basis for the hydrolytic activity of the transpeptidase-like protein DpaA to detach Braun's lipoprotein from peptidoglycan.

IMPORTANCE: Cross-linking reaction of Braun's lipoprotein (Lpp) to peptidoglycan (PG) is catalyzed by some members of the YkuD family of transpeptidases. However, the exact opposite reaction of cleaving the Lpp-PG cross-link is performed by DpaA, which is also a YkuD-like protein. In this work, we determined the crystal structure of DpaA to provide the molecular rationale for the ability of the transpeptidase-like protein to cleave, rather than form, the Lpp-PG linkage. Our findings also revealed the structural features that distinguish the different functional types of the YkuD family enzymes from one another.

A 5+1 assemble-to-activate mechanism of the Lon proteolytic machine.

Many AAA+ (ATPases associated with diverse cellular activities) proteins function as protein or DNA remodelers by threading the substrate through the central pore of their hexameric assemblies. In this ATP-dependent translocating state, the substrate is gripped by the pore loops of the ATPase domains arranged in a universal right-handed spiral staircase organization. However, the process by which a AAA+ protein is activated to adopt this substrate-pore-loop arrangement remains unknown. We show here, using cryo-electron microscopy (cryo-EM), that the activation process of the Lon AAA+ protease may involve a pentameric assembly and a substrate-dependent incorporation of the sixth protomer to form the substrate-pore-loop contacts seen in the translocating state. Based on the structural results, we design truncated monomeric mutants that inhibit Lon activity by binding to the native pentamer and demonstrated that expressing these monomeric mutants in Escherichia coli cells containing functional Lon elicits specific phenotypes associated with lon deficiency, including the inhibition of persister cell formation. These findings uncover a substrate-dependent assembly process for the activation of a AAA+ protein and demonstrate a targeted approach to selectively inhibit its function within cells.

Complete three-dimensional structures of the Lon protease translocating a protein substrate.

Lon is an evolutionarily conserved proteolytic machine carrying out a wide spectrum of biological activities by degrading misfolded damaged proteins and specific cellular substrates. Lon contains a large N-terminal domain and forms a hexameric core of fused adenosine triphosphatase and protease domains. Here, we report two complete structures of Lon engaging a substrate, determined by cryo­electron microscopy to 2.4-angstrom resolution. These structures show a multilayered architecture featuring a tensegrity triangle complex, uniquely constructed by six long N-terminal helices. The interlocked helix triangle is assembled on the top of the hexameric core to spread a web of six globular substrate-binding domains. It serves as a multipurpose platform that controls the access of substrates to the AAA+ ring, provides a ruler-based mechanism for substrate selection, and acts as a pulley device to facilitate unfolding of the translocated substrate. This work provides a complete framework for understanding the structural mechanisms of Lon.

Structural Basis for the Peptidoglycan-Editing Activity of YfiH.

Bacterial cells are encased in peptidoglycan (PG), a polymer of disaccharide N-acetylglucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) cross-linked by peptide stems. PG is synthesized in the cytoplasm as UDP-MurNAc-peptide precursors, of which the amino acid composition of the peptide is unique, with l-Ala added at the first position in most bacteria but with l-Ser or Gly in some bacteria. YfiH is a PG-editing factor whose absence causes misincorporation of l-Ser instead of l-Ala into peptide stems, but its mechanistic function is unknown. Here, we report the crystal structures of substrate-bound and product-bound YfiH, showing that YfiH is a cytoplasmic amidase that controls the incorporation of the correct amino acid to the nucleotide precursors by preferentially cleaving the nucleotide precursor by-product UDP-MurNAc-l-Ser. This work reveals an editing mechanism in the cytoplasmic steps of peptidoglycan biosynthesis. IMPORTANCE YfiH is a peptidoglycan (PG)-editing factor required for the maintenance of specific amino acid compositions of the stem peptides. However, the activity of YfiH has not been deciphered, and the editing mechanism involving YfiH has remained a mystery. Through X-ray crystallographic and biochemical analyses, we demonstrate that YfiH is a hydrolase with a previously unknown activity specific for the UDP-MurNAc-monopeptide, one of the nucleotide precursors from the cytoplasmic steps of the PG biosynthesis pathway. YfiH selectively hydrolyzes UDP-MurNAc-Ser, an incorrect by-product of the biosynthesis reaction, to ensure that only the correct PG precursor, UDP-MurNAc-Ala, is incorporated. Therefore, this work reveals coupled synthetic and editing reactions in the cytoplasmic steps of PG biosynthesis.

A cultural accommodation model for cross-cultural psychotherapy: Illustrated with the case of Asian Americans.

As an extension of F. T. L. Leong's (1996) integrative model, this article presents the cultural accommodation model (CAM), an enhanced theoretical guide to effective cross-cultural clinical practice and research. Whereas F. T. L. Leong's model identifies the importance of integrating the universal, group, and individual dimensions, the CAM takes the next step by providing a theoretical guide to effective psychotherapy with culturally different clients by means of a cultural accommodation process. This model argues for the importance of selecting and applying culture-specific constructs when working with culturally diverse groups. The first step of the CAM is to identify cultural disparities that are often ignored and then accommodate them by using current culturally specific concepts. In this article, several different cultural "gaps" or culture-specific constructs of relevance to Asian Americans with strong scientific foundations are selected and discussed as they pertain to providing effective psychotherapy to this ethnic minority group. Finally, a case study is incorporated to illustrate application of the CAM. (PsycINFO Database Record (c) 2010 APA, all rights reserved).

Intraocular lens power calculation after automated lamellar keratoplasty for high myopia.

PURPOSE: To report intraocular lens (IOL) power calculation in 2 eyes that were highly undercorrected by previous myopic automated lamellar keratoplasty (ALK). METHODS: A 35-year-old man underwent bilateral myopic ALK, which caused high residual myopia (-9.0 -4.0 x 171 and -9.5 -4.5 x 74). The patient then underwent cataract surgery with IOL implantation for cataract development. The double-K clinical history method was utilized, and satisfactory IOL power prediction results were obtained. Two no-history IOL power calculation methods (Rosa correcting factor method and Ferrara theoretical variable refractive index method), which involved axial length-dependent modification of the keratometer-measured corneal radius, and 1 no-history IOL power calculation method (Shammas' method), which involved axial length-independent modification of the keratometer-measured corneal power, were tested on these 2 eyes. RESULTS: In both eyes, the double-K SRK-T clinical history method gave small IOL prediction errors (-0.66 and -0.81 D). The Shammas' no-history method gave a slightly higher IOL prediction error in the right eye (-1.67 D) and a small IOL prediction error in the left eye (-0.74 D). Unacceptable IOL power prediction errors would have resulted if Rosa's correcting factor method (-8.07 and -8.35 D) or Ferrara's theoretical variable refractive index method (-17.56 and -18.51 D) had been applied. When we utilized Rosa's method for the IOL power calculation by assuming that the previous ALK had fully corrected the refractive error, the predicted IOL powers were very close to the benchmark IOL powers (IOL power prediction errors: 1.16 and 0.37 D). When we utilized Ferrara's method with the same assumption, the IOL power prediction errors remained high (-6.32 and -7.16 D). CONCLUSIONS: For patients who have had previous myopic ALK (and whose eyes are highly undercorrected) and who require cataract surgery and for whom the pre-ALK history is available, the double-K method appears to yield excellent predictive results. However, if the pre-ALK history is not available, the Shammas' no-history method appears to yield better results than the Rosa's or the Ferrara's method. High undercorrection by the previous ALK might have been one of the major reasons why Rosa's method resulted in a high IOL prediction error in these 2 eyes. The cause for the marked IOL prediction error by Ferrara's method in this case, however, remains to be determined.