Weak protein-protein interactions (PPIs) (K(D) > 10(-6) M) are critical determinants of many biological processes. However, in contrast to a large growing number of well-characterized, strong PPIs, the weak PPIs, especially those with K(D) > 10(-4) M, are poorly explored. Genome wide, there exist few 3D structures of weak PPIs with K(D) > 10(-4) M, and none with K(D) > 10(-3) M. Here, we report the NMR structure of an extremely weak focal adhesion complex (K(D) approximately 3 x 10(-3) M) between Nck-2 SH3 domain and PINCH-1 LIM4 domain. The structure exhibits a remarkably small and polar interface with distinct binding modes for both SH3 and LIM domains. Such an interface suggests a transient Nck-2/PINCH-1 association process that may trigger rapid focal adhesion turnover during integrin signaling. Genetic rescue experiments demonstrate that this interface is indeed involved in mediating cell shape change and migration. Together, the data provide a molecular basis for an ultraweak PPI in regulating focal adhesion dynamics during integrin signaling.

Clavulanic acid is a potent mechanism-based inhibitor of TEM-1 and SHV-1␤-lactamases,enzymesthatconferresistanceto␤-lactamsinmany Gram-negative pathogens. This compound has enjoyed widespread clinical use as part of ␤-lactam ␤-lactamase inhibitor therapy directed against penicillin-resistant pathogens. Unfortunately, the emergence of clavulanic acid-resistant variants of TEM-1 and SHV-1 ␤-lactamase significantly compromise the efficacy of this combination. A single amino acid change at Ambler position Ser 130 (Ser 3 Gly) results in resistance to inactivation by clavulanate in the SHV-1 and TEM-1 ␤-lactamases. Herein, we investigated the inactivation of SHV-1 and the inhibitor-resistant S130G variant ␤-lactamases by clavulanate. Using liquid chromatography electrospray ionization mass spectrometry, we detected multiple modified proteins when SHV-1 ␤-lactamase is inactivated by clavulanate. Matrixassisted laser desorption ionization-time of flight mass spectrometry was used to study tryptic digests of SHV-1 and S130G ␤-lactamases (؎ inactivation with clavulanate) and identified peptides modified at the active site Ser 70. Ultraviolet (UV) difference spectral studies comparing SHV-1 and S130G ␤-lactamasesinactivatedbyclavulanateshowedthattheformation of reaction intermediates with absorption maxima at 227 and 280 nm are diminished and delayed when S130G ␤-lactamase is inactivated. We conclude that the clavulanic acid inhibition of the S130G variant ␤-lactamase must follow a branch of the normal inactivation pathway. These findings highlight the importance of understanding the intermediates formed in the inactivation process of inhibitor-resistant ␤-lactamases and suggest how strategic chemical design can lead to novel ways to inhibit ␤-lactamases.Among Gram-negative bacteria, ␤-lactamase enzymes (EC 3.5.2.6) are the principal agents of bacterial resistance to penicillin and cephalosporin antibiotics. ␤-Lactamases hydrolyze ␤-lactams and render them ineffective before they reach their targets, the penicillin-binding proteins. This two-step process requires the presence of a strategically located water molecule in the active site (1). To combat the critical problem of ␤-lactamase-mediated resistance, two approaches were undertaken: design ␤-lactams resistant to the hydrolytic action of ␤-lactamases or find inhibitors of these enzymes (2-5). Currently, tazobactam 1, sulbactam 2, and clavulanic acid 3 are the only ␤-lactamase inhibitors used in combination with ␤-lactams for the treatment of infections by bacteria that possess class A ␤-lactamases ( Fig. 1) (6). Administered with a ␤-lactam, ␤-lactamase inhibitors (e.g. ampicillin/ sulbactam, amoxicillin/clavulanate, piperacillin/tazobactam, cefoperazone/sulbactam, and ticarcillin/clavulanate) have had a significant impact on the treatment of a wide variety of infections.TEM-1 and SHV-1 are class A ␤-lactamases commonly found in Escherichia coli and Klebsiella pneumoniae, pathogens responsible for urinary tract, respiratory tract, and bloodstream infections (6). In the...

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Infrared (IR) spectroscopy measures the absorption of infrared radiation by chemical bonds in a material. Chemical structural fragments of molecules, known as functional groups, tend to absorb IR radiation in the same frequency range regardless of the structure of the rest of the molecule that the functional group is in. This correlation between the structure of a molecule and the frequencies at which it absorbs IR radiation allows the structure of unknown molecules to be identified and structural or chemical changes of the molecule to be followed.The clinical applications of IR spectroscopy have been previously reviewed in 1996, covering significant applications of the methodology toward clinical diagnosis and noninvasive, in vivo monitoring from October 1994 to October 1996 (H1). This review covers the Chemical Abstracts period from October 1996 to October 1998. The organization of this contribution is slightly different from that of the previous biennal review. This article will briefly discuss recent advances in the application of IR spectroscopy in diagnostic analyses, laboratory analyses of pathological samples, and noninvasive in vivo monitoring, and examples of each will be presented. Emphasis will be placed on recent advancement of IR spectroscopy employed in the understanding of the process of diseases. Primarily the new technique of IR microspectroscopy will be covered in detail. In addition, basic theory and related data analysis techniques essential to the analysis of multicomponents in biofluids and solid biosamples and essential to the use of IR microspectroscopy in the detection of disease states will also be discussed in detail. REVIEWSThe application of IR spectroscopy to clinical and biomedical analyses continues to increase throughout this review period. There has been a number of representative reviews and books published (H2-H11). Different infrared measurement techniques in the clinical analysis of biofluids were reviewed by Wang et al. (H3). The authors presented the spectra of physiological samples measured as liquids and thin films using transmission, attenuated total reflection, photoacoustic, and diffuse reflectance IR spectroscopy. They described the advantages and limitations of each of these techniques and discussed the development of routine analysis methods for biofluids. Three books have been published on spectroscopic methods for studies of biomolecules and specifically for IR methods including IR microscopy (H2, H4, H6). Jackson et al. reviewed IR spectroscopy as a new frontier in medicine (H7). They included 53 references on IR methodologies which comprise both instrumental (imaging and spatially localized IR spectroscopy) and interpretational procedures aimed at optimizing the measurements and their conversion to biodiagnostic information. A review with 15 references presents the great power of FT-IR spectroscopy in the field of medicinal biology (H8).Rintoul et al. demonstrated in their review article the versatility of modern FT-IR spectrometry by describing the range of so...

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